Anti FGF23 antibody and a pharmaceutical composition comprising the same

ABSTRACT

To provide an antibody against FGF23 and a pharmaceutical composition such as a preventive or therapeutic agent which can prevent or treat by suppressing an action of FGF23 by using the antibody. An antibody or its functional fragment against human FGF23 produced by hybridoma C10 (Accession No. FERM BP-10772).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.12/030,593 filed Feb. 13, 2008, which claims priority from JapaneseApplication No. 2007-034018 filed Feb. 14, 2007. The subject matter ofeach of the above-referenced applications is incorporated in entirety byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an anti-FGF23 antibody whichspecifically binds to an FGF23 antigen. Furthermore, the presentinvention relates to an agent for prevention or treatment of mineralmetabolic disorders due to excessive production of FGF23 or other causescomprising as an active ingredient the anti-FGF23 antibody. Inparticular, the present invention relates to an agent for treatment ofhypophosphatemic rickets and osteomalachia treatment agent.

2. Background Art

Fibroblast growth factor was first purified from a bovine pituitarygland as a substance that stimulates an increase in fibroblast cell lineNIH3T3. Since then, similar proteins have been identified varioustissues, and a group of the substances compose a polypeptide family (FGFfamily). Up until now, 22 proteins have been identified in vertebratesas belonging to the FGF family. With regard to the biological activityof these proteins, not only do they have fibroblast growth activity, butthese proteins are also known to have divergent actions such as growthof the mesoblast and the neuroectoderm, and angiogenesis action, andlimb bud formation in the developmental stage. FGF is also varied in thegene expression site and expression time. They are often expressed onlyat certain sites only in the developmental stage or in adults. At least4 genes encoding the FGF receptor are known, FGFR1, FGFR2, FGFR3, andFGFR4. In addition, with regards to FGFR1, FGFR2, and FGFR3, it is knownthat there are receptor proteins for each with differing extracellulardomains due to differences in splicing. In addition, heparin and heparansulfate proteoglycan are known to control the action by interaction withFGF and FGF receptors. In addition, there are many, which, due tostructural similarities, belong to the FGF family, but whose biologicalactivities and receptor binding properties and the like have not beenknown. The characteristics of this FGF family have been summarized in areview (see Ornitz, D. et al., Genome biology, 2: 3005.1-3005.12, 2001).

FGF23 (in general, may also be represented as FGF-23) was clonedinitially from a mouse by a database search using homology with FGF15and the PCR method. Further, human FGF23 was cloned by using thesequence homology with mouse FGF23. Human FGF23 is a polypeptide with251 amino acid residues. In addition, as the secretory signal sequence,an amino acid sequence at amino terminal side up to 24 amino acids ispredicted to be cleaved at the time of secretion (see Yamashita, T. etal., Biochem. Biophy. Res. Commun., 277: 494-498, 2000). Next, inresearch on autosomal dominant hypophosphatemic rickets/osteomalachia(henceforth referred to as ADHR), the mutated gene region in ADHRpatients was narrowed down and with advancement in the identification ofthe responsible gene, a mis-sense mutation in the FGF23 gene wasdiscovered characteristically in ADHR patients (see White, K. E. et al.,Nature Genet., 26: 345-348, 2000). With this discovery, there was astrong suggestion that FGF23 was physiologically important in the body.On the other hand, what determined the biological activity of FGF23 wasresearch into neoplastic osteomalachia which is one of thehypophosphatemia rickets and osteomalachia diseases. In this disease,the culprit neoplasm of the disease produces and secretes a liquiddisease initiating factor, and it is thought that pathologies such ashypophosphatemia, osteomalachia and the like are caused by the diseaseinitiating factor.

In the search for the disease initiating factor produced by this culpritneoplasm, FGF23 was cloned as a gene which is overexpressed in thetumor. Furthermore, by administering this factor, it was shown thathypophosphatemia and osteomalachia were reproduced (see Shimada, T. etal., Proc. Natl. Acad. Sci., 98: 6500-6505, 2001 and InternationalPublication Number WO02/14504 pamphlet). Based on this research, FGF23has been shown to be related in the metabolic control related tophosphorus and calcium in the body. In addition, it was suggested thatthis acts as a systemic factor which expresses its action by circulatingin the body. Furthermore, later research also showed that the blood ofactual neoplastic osteomalachia patients had a higher value of FGF23concentration as compared to healthy subjects (see Yamazaki, Y. et al.,J. Clin. Endocrinol. Metab., 87: 4957-4960, 2002 and Jonsson, K. B., etal., N. Engl. J. Med., 348: 1656-1663, 2003).

In addition, X-linked hypophosphatemic rickets (henceforth referred toas XLH) is a disease which is known to have a similar presentation asADHR and neoplastic osteomalachia in terms of clinical findings. In thisdisease as well, the FGF23 concentration in the blood was shown to be ata high value (see Yamazaki, Y. et al., J. Clin. Endocrinol. Metab., 87:4957-4960, 2002 and Jonsson, K. B., et al., N. Engl. J. Med., 348:1656-1663, 2003).

In other words, the cause for vitamin D resistant rickets andosteomalachia which were observed in neoplastic osteomalachia, XLH, andthe like had been previously unknown, but the secreted disease causingfactor was shown to be FGF23. Furthermore, with regard to other mineralmetabolic diseases such as fibrous dysplasia, McCune-Albright syndrome,autosomal recessive hypophosphatemia rickets, and the like, highconcentrations of FGF23 in the blood have been reported to be associatedwith hypophosphatemia and rickets and osteomalachia (See Riminucci, M.et al., J. Clin. Invest., 112: 683-692, 2003; Yamamoto, T. et al., J.Bone Miner. Metab., 23: 231-237, 2005; Lorenz-Depiereux, B. et al., Nat.Genet., 38: 1248-1250, 2006).

From the above report, the condition of having excessive FGF23 in thebody has been shown to induce hypophosphatemia and the accompanyingrickets and osteomalachia and the like. Furthermore, for chronic renalinsufficiency hyperphosphatemia, abnormally high serum FGF23 values havebeen reported. Excessive FGF23 has been suggested to be possibly relatedto a portion of the mineral metabolic diseases during renalinsufficiency (see Gupta, A. et al., J. Clin. Endocrinol. Metab., 89:4489-4492, 2004 and Larsson, T. et al., Kidney Int., 64: 2272-2279,2003). With regard to these diseases induced due to excessive FGF23,suppressing the action of FGF23 or removing FGF23 is thought to be apossible way to treat the diseases. Up to now, anti-FGF23 mousemonoclonal antibody has been reported to be a way to suppress the actionof FGF23 (see Yamashita, T. et al., Biochem. Biophy. Res. Commun., 277:494-498, 2000). When the anti-FGF23 mouse monoclonal antibody 2C3B and3C1E used in this report were administered to normal mice, the functionof the endogenous mouse FGF23 was inhibited, and the phosphorusexcretion from the kidney was suppressed. By fluctuating the expressionof vitamin D-metabolizing enzyme in the kidney, this was shown to resultin increased concentrations for phosphorus and 1α, 25 dihydroxy vitaminD (henceforth referred to as 1,25D) in the serum. Furthermore, repeatedadministration of anti-FGF23 mouse monoclonal antibody was conducted onHyp mouse which is a model mouse for XLH which has a high serumconcentration of FGF23 and has hypophosphatemia and has bone elongationdysfunction and calcification dysfunction. As a result, in the Hyp mice,a rise in the phosphorus concentration in the blood was seen, and inaddition, there were improvements in bone elongation dysfunction andcalcification dysfunction. From these results, the use of FGF23 actionsuppressing antibody was thought to be appropriate as a medicine forFGF23 excess diseases. However, the 2C3B and 3C1E antibodies used inthis report are mouse-derived antibodies. Mouse antibodies which arerecognized as foreign by human host initiates a so-called “humananti-mouse antibody” (in other words HAMA) response, and there may besituations where serious side-effects are seen (see Van Kroonenbergh, M.J. et al., Nucl. Med. Commun. 9: 919-930, 1988).

In order to avoid this type of problem, one approach was to develop achimera antibody (see European Patent Application Publication Number120694 Specification and European Patent Application Publication Number125023 Specification). Chimera antibodies include a portion of antibodyderived from 2 or more species (for example, variable region of themouse antibody and the constant region of the human antibody and thelike). The advantage of this type of chimera antibody is that thebinding to the antibody which was the characteristic of the originalmouse antibody is maintained, but on the other hand, “a human-antichimera antibody” (in other words “HACA”) response is still induced (seeBruggemann, M. et al., J. Exp. Med., 170: 2153-2157, 1989).

Furthermore, a recombinant antibody has been developed where only aportion of the substituted antibody is a complementarity determiningregion (CDR) (see British Patent Number GB2188638A specification andU.S. Pat. No. 5,585,089 specification). Using CDR transplant technology,an antibody consisting of mouse CDR, the framework of the human variableregion and constant region (in other words “humanized antibody”) wasproduced (see Riechmann, L. et al., Nature, 332: 323-327, 1988). It hasbeen known that using this method, anti-FGF23 mouse antibody such as2C3B antibody can be humanized by substituting mouse antibody with ahuman antibody sequence. However, when humanized, there is thepossibility that the affinity to the antigen may be reduced. Inaddition, for the current treatment of hypophosphatemia rickets in XLHand the like, the main method is periodic oral administration of VitaminD formulation and phosphoric acid. However, there is the problem thatthe patients are forced to have a substantial burden due to the size ofeach dose and the dosage frequency per day. Therefore, in order tolessen the burden on the patients and their families, a hypophosphatemiatreatment drug which shows a sustained raising action for serumphosphate concentration and serum 1,25D concentration is desired inorder to extend the time between doses.

SUMMARY OF THE INVENTION

The object of the present invention is to provide human antibody againstFGF23 and to provide a pharmaceutical composition such as an agent forprevention or treatment or the like with few side effects by using theantibody to suppress the action of FGF23 and thereby preventing ortreating disease.

Furthermore, the object of the present invention is to provide anantibody which is an anti-FGF23 antibody which can be used as ahypophosphatemia treatment medicine having a more sustained raisingaction for serum phosphate concentration and serum 1,25D concentrationwith a single dose as compared to existing anti-FGF23 antibodies.Another object of the present invention is to provide a pharmaceuticalcomposition such as an agent for prevention or treatment of a diseaserelated to FGF23 using this antibody.

Currently, the mainstream treatment method for hypophosphatemia ricketsis oral administration of vitamin D formulation together with phosphateperiodically several times a day. However, because of the large amountof each dose and the frequency of doses per day, there is the problemthat the patients are forced to have a large burden. The anti-FGF23human monoclonal antibody, the C10 antibody, obtained by the presentinvention is shown to have a more sustained raising action for the bloodphosphate concentration and 1,25D concentration, in other words, astronger FGF23 neutralizing activity. With a single administration ofthe C10 antibody in the present research, there was observed a sustainedraising action for serum phosphate concentration and serum 1,25Dconcentration. This suggests that as compared to the current treatmentfor hypophosphatemia, the C10 antibody has the potential for being adramatically superior treatment.

The present invention is as follows.

[1] An antibody against human FGF23 or a functional fragment thereof,comprising a heavy chain variable region and/or a light chain variableregion of an antibody produced by hybridoma C10 (Accession No. FERMBP-10772)

[2] An antibody against human FGF23 or a functional fragment thereof,comprising a heavy chain amino acid sequence shown by an amino acidsequence from Q at position 20 to S at position 136 of SEQ ID NO: 12and/or a light chain amino acid sequence shown by an amino acid sequencefrom A at position 23 to K at position 128 of SEQ ID NO: 14.

[3] An antibody against human FGF23 or a functional fragment thereof,wherein: the antibody against human FGF23 or the functional fragmentthereof contains a heavy chain variable region and/or a light chainvariable region amino acid sequence; and the heavy chain variable regionamino acid sequence is shown by an amino acid sequence from Q atposition 20 to S at position 136 of SEQ ID NO: 12; and the light chainvariable region amino acid sequence is shown by an amino acid sequencefrom A at position 23 to K at position 128 of SEQ ID NO: 14.

[4] An antibody against human FGF23 produced by hybridoma C10 (AccessionNo. FERM BP-10772) or a functional fragment thereof.

[5] An antibody or a functional fragment thereof binding to all or partof epitope on human FGF23, to which an antibody produced by hybridomaC10 (Accession No. FERM BP-10772) binds.

[6] The antibody against human FGF23 or a functional fragment thereof,comprising a heavy chain variable region of the above [3] having any oneof complementarity determining region (CDR) 1 shown by the amino acidsequence of SEQ ID NO: 40, CDR2 shown by the amino acid sequence of SEQID NO: 41 and CDR3 shown by the amino acid sequence of SEQ ID NO: 42, ora heavy chain variable region of the above [3] having all of the above.

[7] The antibody against human FGF23 or a functional fragment thereof,comprising a light chain variable region of the above [3] having any oneof CDR 1 shown by the amino acid sequence of SEQ ID NO: 43, CDR2 shownby the amino acid sequence of SEQ ID NO: 44 and CDR3 shown by the aminoacid sequence of SEQ ID NO: 45, or a light chain variable region of theabove [3] having all of the above.

[8] An antibody against human FGF23 or a functional fragment thereof,wherein the antibody against human FGF23 or the functional fragmentthereof contains a heavy chain variable region having any one ofcomplementarity determining region (CDR) 1 shown by the amino acidsequence of SEQ ID NO: 40, CDR2 shown by the amino acid sequence of SEQID NO: 41 and CDR3 shown by the amino acid sequence of SEQ ID NO: 42, ora heavy chain variable region having all of the above; and a light chainvariable region having any one of complementarity determining region(CDR) 1 shown by the amino acid sequence of SEQ ID NO: 43, CDR2 shown bythe amino acid sequence of SEQ ID NO: 44 and CDR3 shown by the aminoacid sequence of SEQ ID NO: 45, or a light chain variable region havingall of the above.

[9] The antibody against human FGF23 or a functional fragment thereof asdescribed in any one of [1]-[8], wherein the functional fragment is apeptide fragment selected from the group consisting of Fab, Fab′, F(ab′)2, disulfide stabilized Fv (dsFv), dimerized V region (diabody),single chain Fv (scFv) and CDR.

[10] The antibody against human FGF23 or a functional fragment thereof,as described in any one of [1]-[8], comprising: a heavy chain and/orlight chain having an amino acid sequence in which one or several aminoacids are deleted, substituted or added.

[11] The antibody against human FGF23 as described in any one of[1]-[10], wherein the class of the antibody is IgG, IgA, IgE, or IgM.

[12] The antibody against human FGF23 as described in [11], wherein thesubclass of the antibody is IgG1, IgG2, IgG3, or IgG4.

[13] A pharmaceutical composition, comprising as an active ingredient,the antibody against human FGF23 or a functional fragment thereof asdescribed in any one of [1]-[12].

[14] A pharmaceutical composition which can control phosphorusmetabolism and/or vitamin D metabolism by FGF23 and comprises, as anactive ingredient, the antibody against human FGF23 or a functionalfragment thereof as described in any one of [1]-[12].

[15] A pharmaceutical composition for prevention or treatment ofdiseases that are associated with mineral metabolism disorderscomprising as an active ingredient, the antibody against human FGF23 ora functional fragment thereof as described in any one of [1]-[12].

[16] The pharmaceutical composition as described in [15], wherein thedisease which is associated with mineral metabolism abnormalities isselected from the group consisting of neoplastic osteomalachia, ADHR,XLH, fibrous dysplasia, McCune-Albright syndrome, and autosomalrecessive hypophosphatemia.

[17] A pharmaceutical composition for prevention or treatment of adisease selected from the group consisting of osteoporosis, rickets,hypocalcaemia, hypocalcaemia, heterotrophic calcification,osteosclerosis, Paget's disease, hyperparathyroidism,hypoparathyroidism, and pruritis, comprising as an active ingredient,the antibody against human FGF23 or a functional fragment thereof asdescribed in any one of [1]-[12].

[18] A hybridoma C10 (Accession No. FERM BP-10772).

[19] Nucleic acids which encode an amino acid sequence of a heavy chainvariable region encoded by a base sequence from C at position 58 to A atposition 408 represented by SEQ ID NO: 11.

[20] Nucleic acids which encode an amino acid sequence of a light chainvariable region encoded by a base sequence from G at position 67 to A atposition 384 represented by SEQ ID NO: 13.

[21] A vector containing the nucleic acid described in [19] or [20].

[22] A host cell containing the vector described in [21].

[23] A method for producing an antibody against human FGF23 or afunctional fragment thereof, comprising the step of culturing the hostcell described in [22] to express an antibody against human FGF23 or afunctional fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the construction steps of C10expression vector.

FIG. 2 shows the nucleotide sequence (SEQ ID NO: 30) and amino acidsequence (SEQ ID NO: 31) of the antibody heavy chain gene inN5KG1_C10_LH. The amino acid sequence surrounded by a rectangular linerepresents the secretion signal sequence (leader sequence).

FIG. 3 shows the nucleotide sequence (SEQ ID NO: 32) and amino acidsequence (SEQ ID NO: 33) of the antibody light chain gene inN5KG1_C10_LH. The amino acid sequence surrounded by a rectangular linerepresents the secretion signal sequence (leader sequence).

FIG. 4 shows the structure of C10 expression vector.

FIG. 5A shows the result of the measurement for detecting purified fulllength human FGF23 protein by the sandwich ELISA method using 2C3Bantibody or C10 antibody as the immobilizes antibody, and 3C1E antibodyas the detection antibody.

FIG. 5B shows the result of the measurement for detecting the culturesupernatant of cynomolgus monkey FGF23 expressing cells by the sandwichELISA method using 2C3B antibody or C10 antibody as the solid phaseantibody, and 3C1E antibody as the detection antibody.

FIG. 6 is a graph showing chronological measurements of serumphosphorous concentration in cynomolgus monkeys administered withsolvent, 2C3B antibody or C10 antibody. Measured values are shown inaverage+/−standard error. Further, when significant difference testbetween the solvent administered group and the test groups was conductedat the same date using Student-test, values found to be significantdifference (p<0.05) are marked with * on the graph.

FIG. 7 is a graph showing an increase in the serum phosphorousconcentration of cynomolgus monkeys 5 days after 2C3B antibody or C10antibody administration, based on the serum phosphorous concentration ofcynomolgus monkey 5 days after the administration of the solvent as thestandard.

FIG. 8 is a graph showing chronological measurements of serum 1,25Dconcentration in cynomolgus monkeys administered with solvent, 2C3Bantibody or C10 antibody. Measured values are shown inaverage+/−standard error. Further, when significant difference testbetween the solvent administered group and the test groups was conductedat the same date using Student-test, values found to be significantdifference (p<0.05) are marked with * on the graph.

FIG. 9 is a picture showing the detection of the culture supernatant ofthe cells with no forced expression (control) and the culturesupernatant of the human and cynomolgus monkey FGF23 expression cells byC15 antibody using the Western blotting method.

FIG. 10 shows the structure of pPSs FGF23 vector.

FIG. 11 shows the structure of pUS FGF23 KI vector.

FIG. 12 represents an allele structure in which the drug resistance gene(loxp-neor) is targeted, an allele structure in which human FGF23(−SP)+drug resistance gene (loxpv-puror) is targeted by using pUS hFGF23KI vector, an allele structure in which the drug resistance genes(loxp-neor, loxpv-puror) are deleted, and the position of Southernanalysis probe. Terms used in the figures are described in detail asfollows:

-   -   hFGF23 (−SP): Human FGF23 gene having no specific signal peptide        code region,    -   Cκ: constant region of mouse Igκ gene,    -   loxpv-puro: puromycin resistance gene having loxPV sequence        which is a partially mutated loxP sequence at both ends thereof,    -   loxp-neor: neomycin resistance gene having loxP sequence at both        ends thereof, Ck3′ probe: Southern blotting analysis probe for        selection of clones having hFGF23 (−SP)+loxpv-puror gene        introduced and having loxpv-puror gene deleted,    -   3′KO-probe: Southern blotting analysis probe for selection of        clones having loxp-neor gene introduced and deleted, and    -   E: EcoRI restriction enzyme site.

FIG. 13 is a graph showing the serum FGF23 concentration 7 days beforethe control antibody or C10 antibody administration. Measured values areshown in average+/−standard error. Further, when significant differencetest between the WT mice group and the test groups was conducted usingStudent's t-test, groups found to be significant difference (p<0.001)are marked with *** on the graph.

FIG. 14 is a graph showing the serum phosphorous concentration 7 daysbefore the control antibody or C10 antibody administration and 3 daysafter the first administration of control antibody or C10 antibody.Measured values are shown in average+/−standard error. Further, whensignificant difference test between the WT mice group and the testgroups was conducted in one day using Student's t-test, groups found tobe significant difference (p<0.001) are marked with *** on the graph. Inaddition, when significant difference test between the hFGF23KI mousecontrol antibody administered group and the test groups was conducted inone day, hFGF23KI mouse C10 antibody administered groups found to besignificant difference (p<0.001) are marked with ### on the graph.

FIG. 15 is a graph showing the serum phosphorous concentration 1 dayafter the fifth administration of control antibody or C10 antibody.Measured values are shown in average+/−standard error. Further, whensignificant difference test between the WT mice group and the testgroups was conducted using Student's t-test, groups found to besignificant difference (p<0.001) are marked with *** on the graph. Inaddition, when significant difference test between the hFGF23KI mousecontrol antibody administered group and the test groups was conducted,hFGF23KI mouse C10 antibody administered groups found to be significantdifference (p<0.001) are marked with ### on the graph.

FIG. 16 is a graph showing the grip strength 1 day after the fourthadministration of control antibody or C10 antibody. Measured values areshown in average+/−standard error. Further, when significant differencetest between the WT mice group and the test groups was conducted usingStudent's t-test, groups found to be significant difference (p<0.001)are marked with *** on the graph. In addition, when significantdifference test between the hFGF23KI mouse control antibody administeredgroup and the test groups was conducted, hFGF23KI mouse C10 antibodyadministered groups found to be significant difference (p<0.001) aremarked with ### on the graph.

FIG. 17 is a picture showing the histological staining image of femurcollected from mice 1 day after the fifth administration of controlantibody or C10 antibody, wherein the staining was performed byVillanueva-Goldner method.

FIG. 18 is a graph showing the ratio of ash weight to dry weight oftibia collected from mice 1 day after the fifth administration ofcontrol antibody or C10 antibody. Measured values are shown inaverage+/−standard error. Further, when significant difference testbetween the WT mice group and the test groups was conducted usingStudent's t-test, groups found to be significant difference (p<0.001)are marked with *** on the graph. In addition, when significantdifference test between the hFGF23KI mouse control antibody administeredgroup and the test groups was conducted, hFGF23KI mouse C10 antibodyadministered groups found to be significant difference (p<0.001) aremarked with ### on the graph.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Below, by clarifying the definitions for the terms used in the presentinvention, we will describe the present invention in detail.

I. Antibody of the Present Invention

1. Anti-FGF23 Antibody and its Functional Fragment

The antibody of the present invention is an antibody against FGF23 whichis a member of the fibroblast growth factor (FGF) family.

In the present invention, “antibody against FGF23” is an antibody whichbinds to FGF23 or a portion thereof, an antibody which is reactive toFGF23 or a portion thereof, or an antibody which recognizes FGF23 or aportion thereof. Antibody against FGF23 is also termed an anti-FGF23antibody. In the present invention, an antibody is an immunoglobulin inwhich all of the regions which construct the immunoglobulin of the heavychain variable region and heavy chain constant region and the lightchain variable region and light chain constant region are derived from agene which encodes the immunoglobulin. The antibody is preferably amonoclonal antibody. Here, a portion of FGF23 signifies a partial aminoacid sequence of a full-length amino acid sequence of FGF23 representedby SEQ ID NO: 4 and is a fragment peptide of FGF 23 comprising acontinuous amino acid sequence. Preferably, the antibody contains theamino acid sequence from Q at position 20 to S at position 136 of SEQ IDNO: 12 and/or the amino acid sequence from A at position 23 to K atposition 128 of SEQ ID NO: 14. More preferably, the antibody is anantibody produced by hybridoma C10. SEQ ID NO: 12 is the amino acidsequence that comprises the leader sequence of the heavy chain variableregion of the antibody against FGF23. The amino acid sequence from Q atposition 20 to S of number 136 of SEQ ID NO: 12 is the mature portion ofthe amino acid sequence with the leader sequence portion removed. Inaddition, SEQ ID NO: 14 is the amino acid sequence that comprises theleader sequence of the light chain variable region of the antibodyagainst FGF23. The amino acid sequence from A at position 23 to K atposition 128 of SEQ ID NO: 14 is the mature portion of the amino acidsequence with the leader sequence removed. With regard to the class ofantibody, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulinE (IgE), and immunoglobulin M (IgM) are used. Preferably, it is IgG.Furthermore, for the IgG subclass, IgG1, IgG2, IgG3, IgG4 are used. Itis preferably IgG1, IgG2, and IgG4. More preferably, it is IgG1.

The antibody of the present invention also includes an anti-FGF23antibody which comprises an amino acid sequence of a novelcomplementarity determining region (CDR).

A CDR is present in the variable region of an antibody, and the part isresponsible for the specificity of antigen recognition. The part otherthan the CDR in the variable region has a role in maintaining thestructure of the CDR, and is referred to as the framework region (FR). Aconstant region is present in the C terminal side of a heavy chain and alight chain, and is referred to as the heavy chain constant region (CH)and the light chain constant region (CL), respectively.

Three complementarity determining regions are present in the heavy chainvariable region, which are a first complementarity determining region(CDR1), a second complementarity determining region (CDR2), and a thirdcomplementarity determining region (CDR3). The three complementaritydetermining regions in the heavy chain variable region are collectivelyreferred to as the heavy chain complementarity determining region.Similarly, three complementarity determining regions are present in thelight chain variable region, which are a first complementaritydetermining region (CDR1), a second complementarity determining region(CDR2), and a third complementarity determining region (CDR3). The threecomplementarity determining regions in the light chain variable regionare collectively referred to as the light chain complementaritydetermining region. The sequences of these CDRs can be determined byusing the methods described in Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services (1991) and the like.

The antibody of the present invention preferably has at least any one orall of CDR1 shown by SEQ ID NO: 40, CDR2 shown by SEQ ID NO: 41, andCDR3 shown by SEQ ID NO: 42 as the heavy chain complementaritydetermining region. In addition, the antibody of the present inventionpreferably has at least any one or all of CDR1 shown by SEQ ID NO: 43,CDR2 shown by SEQ ID NO: 44, and CDR3 shown by SEQ ID NO: 45 as thelight chain complementarity determining region. More preferably, theantibody of the present invention is an antibody which binds to FGF23and has CDR1 shown by SEQ ID NO: 40, CDR2 shown by SEQ ID NO: 41, andCDR3 shown by SEQ ID NO: 42 as the heavy chain complementaritydetermining region, and CDR1 shown by SEQ ID NO: 43, CDR2 shown by SEQID NO: 44, and CDR3 shown by SEQ ID NO: 45 as the light chaincomplementarity determining region.

The CDR sequence of the antibody of the present invention is notspecifically limited. However, the antibody of the present invention isan antibody preferably comprising any one or more CDRs, more preferablythree CDRs of the heavy chain, and even more preferably six CDRs of theCDR sequences represented by SEQ ID NO: 40 through 45. The amino acidsequence other than the CDR is not specifically limited. The antibody ofthe present invention includes so called CDR transplantation antibodies,wherein the amino acid sequence other than the CDR is derived from otherantibodies, and particularly antibodies in other species. Among these, ahumanized antibody or human antibody, wherein the amino acid sequenceother than the CDR is derived from human, is preferred. An addition,deletion, substitution and/or insertion of 1 amino acid residue or morecan be introduced into the FR according to need. A publicly known methodcan be applied as the method for producing a humanized antibody or humanantibody.

“Functional fragment” is a portion of an antibody (partial fragment) andhas one or more of the actions of the antibody to the antigen. In otherwords, it refers to a fragment which retains binding ability to theantigen, reactivity to the antigen, or recognition capability to theantigen. Examples include Fv, disulfide stabilized Fv (dsFv), singlechain Fv (scFv), and polymers of these and the like. Stated morespecifically, examples include peptides which contain Fab, Fab′, F(ab′)₂, scFv, diabody, dsFv, and CDR [D. J. King., Applications andEngineering of Monoclonal Antibodies., 1998 T. J. International Ltd].

Of the fragments which are obtained by treating an antibody which bindsto FGF23 with the protease papain, Fab is the antibody fragment ofmolecular weight approximately 50,000 with antigen binding activity, inwhich approximately half of the amino-terminal side of the H chain withall of the L chain by a disulfide bond.

The Fab of the present invention can be obtained by treating theantibody which binds to FGF23 with the protease papain. Alternatively,Fab can be produced by inserting DNA which encodes Fab of the antibodyinto an expression vector for prokaryotic organisms or an expressionvector for eukaryotic organisms and expressing this vector byintroducing into a prokaryotic organism or eukaryotic organism.

Of the fragments obtained by treating IgG with the protease pepsin, F(ab′)₂ is the antibody fragment of molecular weight approximately100,000 with antigen binding activity and which is larger than that ofFab bonded via disulfide bonds of the hinge region.

The F (ab′)₂ of the present invention can be obtained by treatingantibody that binds with FGF23 with the protease pepsin. Alternatively,it can be produced through a thioether bond or disulfide bond of Fab′described below.

Fab′ is an antibody fragment of a molecular weight of approximately50,000 having antigen binding activity and in which the disulfide bondof the hinge region of the above F (ab′)₂ is cleaved.

Fab′ of the present invention is obtained by treating F (ab′)₂ of thepresent invention, which binds to FGF23, with a reducing agentdithiothreitol. Alternatively, DNA which encodes the Fab′ fragment ofthis antibody is inserted into an expression vector for prokaryoticorganisms or into an expression vector for eukaryotic organisms, andthis vector is introduced into prokaryotic organisms or eukaryoticorganisms and thereby is expressed to produce Fab′.

scFv is an antibody fragment having antibody binding activity with asingle heavy chain variable region (hereinafter referred to as VH) and asingle light chain variable region (henceforth written as VL) which arelinked using a suitable peptide linker (henceforth written as P) and isa VH-P-VL or VL-P-VH polypeptide.

The scFv of the present invention can be produced by obtaining the cDNAwhich encodes VH and VL of the antibody of the present invention whichbinds with FGF23 and constructing the DNA which encodes scFV andinserting the DNA into the expression vector for prokaryotic organismsor the expression vector for eukaryotic organisms and introducing andexpressing the expression vector in prokaryotic organisms or eukaryoticorganisms.

A diabody is an antibody fragment in which scFv is dimerized and is anantibody fragment having a bivalent antibody binding activity. Eachbinding activity of the bivalent antibody can be the same or different.

The diabody of the present invention can be produced by obtaining thecDNA which encodes the VH and VL of the antibody of the presentinvention which binds to FGF23, constructing the DNA which encodes scFVsuch that the length of the amino acid sequence for the peptide linkeris 8 residues or less, inserting this DNA into an expression vector forprokaryotic organism or expression vector for eukaryotic organism, andexpressing this expression vector by introducing into a prokaryoticorganism or eukaryotic organism.

In dsFv, 1 amino acid residue in each of VH and VL is substituted with acystine residue, and the polypeptides are bonded through a disulfidebond between these cysteine residues. The amino acid residue which issubstituted with the cysteine residue can be selected based on thepredicted tertiary structure of the antibody according to the methodindicated by Reiter et al (Protein Engineering, 7: 697-704, 1994).

The dsFv of the present invention can be produced by obtaining the cDNAwhich encodes VH and VL of the antibody of the present invention whichbinds to FGF23, constructing the DNA which encodes the dsFv, insertingthis DNA into an expression vector for a prokaryotic organism or anexpression vector for a eukaryotic organism, and introducing andexpressing this expression vector in a prokaryotic organism oreukaryotic organism.

The peptide which comprises CDR is constructed comprising at least 1region or more of CDR of VH or VL. Peptides which comprise multipleCDR's can be linked together directly or through a suitable peptidelinker.

The peptide which comprises the CDR of the present invention can beproduced by constructing a DNA which encodes the CDR of the VH and VL ofthe antibody of the present invention which binds to FGF23, insertingthis DNA into an expression vector for prokaryotic organisms orexpression vector for eukaryotic organisms, and introducing andexpressing this expression vector in prokaryotic organisms or eukaryoticorganisms.

In addition, the peptide which contains CDR can be produce by a chemicalsynthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method)and tBoc method (t-butyloxycarbonyl method) and the like.

Furthermore, “functional fragment” is a fragment of the antibody whichcan bind to the antigen (FGF23). Preferably, the “functional fragment”is a fragment which can bind to FGF23 and comprises an amino acidsequence from Q at position 20 to S at position 136 of SEQ ID NO: 12,and/or an amino acid sequence from A at position 23 to K at position 128of SEQ ID NO: 14. Preferably, the “functional fragment” is a fragmentwhich comprises at least one or all of CDRs represented by SEQ ID NO: 40through 45 and can bind to FGF23. More preferably, the “functionalfragment” is derived from the variable region of an antibody produced byhybridoma C10 and is a fragment which can bind to FGF23.

The antibody of the present invention includes derivatives of theantibody in which radioisotopes, low molecular weight drugs,macromolecular drugs, proteins, and the like is bound chemically orthrough genetic engineering to the antibody against FGF23 of the presentinvention or functional fragments of the antibody.

The derivatives of the antibody of the present invention can be producedby bonding radioisotopes, low molecular weight drugs, macromoleculardrugs, proteins and the like to the amino terminal side or carboxyterminal side of the H chain (heavy chain) or L chain (light chain) ofthe antibody against FGF23 of the present invention or the functionalfragment of the antibody, to a suitable substituted group or side chainin the antibody or functional fragment of the antibody, and further, toa sugar chain in the antibody or functional fragment of the antibody andthe like by chemical methods (Koutai Kogaku Nyuumon, Osamu Kanamitsu,Chijin Shokan, 1994) and the like.

In addition, the derivative of the antibody bonded with protein isproduced by linking the DNA which encodes the antibody against FGF23 ofthe present invention and the functional fragment of the antibody andthe DNA which encodes the protein to be bonded, inserting this DNA intoan expression vector, and introducing and expressing the expressionvector in a suitable host cell.

For the radioisotope, examples include 131I, 125I. For example, theradioisotope can be bonded to the antibody by the chloramine T methodand the like.

Low molecular weight drugs include alkylating agents including nitrogenmustard, cyclophosphamide; antimetabolites such as 5-fluorouracil andmethotrexate; antibiotics such as daunomycin, bleomycin, mitomycin C,daunorubicin and doxorubicin; plant alkaloids, such as vincristine,vinblastine and vindesine; anti cancer agents such as hormone agentssuch as tamoxifen and dexamethasone (Clinical oncology; JapaneseClinical Oncology Research Meeting, Japanese Journal of Cancer andChemotherapy Co., 1996); steroids such as hydrocortisone, prednisone,and the like; non-steroid agents including aspirin and indomethacin;immunomodulators such as gold thiomalate, penicillamine, and the like;immunosuppressors such as cyclophosphamide, azathioprine, and the like;anti-inflammatories such as anti-histamines such as chlorpheniraminemaleate, clemastine, and the like (Inflammation and anti-inflammatorytreatment method, Ishiyaku Publishing Corp. Ltd., 1982). The bonding ofthe antibody with these low molecular weight drugs is conducted by knownmethods. Examples of methods for bonding daunomycin with antibodyinclude a method for bonding between amino groups of the daunomycin andantibody via glutaraldehyde, and a method for bonding the amino group ofdaunomycin and carboxyl group of the antibody via water-solublecarbodiimide. By bonding these low molecular weight drugs with theantibody, a derivative of an antibody having the function of the lowmolecular weight drug is obtained.

For the macromolecular drug, examples include polyethylene glycol(hereinafter referred to as PEG), albumin, dextran, polyoxyethylene,styrene maleate copolymer, polyvinyl pyrrolidone, pyran copolymer,hydroxypropyl methacrylamide, and the like. By bonding thesemacromolecular compounds with antibody or a functional fragment of anantibody, the following effects are anticipated (1) the stability withrespect to various chemical, physical, and biological factors isimproved (2) half life in blood is dramatically extended, (3)immunogenicity is lost, antibody production is suppressed, and the like(Bioconjugate Pharmaceutical, Hirokawa Shoten, 1993). An example of amethod for bonding PEG to an antibody is a method of reacting withPEG-modifying reagent (Bioconjugate Pharmaceutical, Hirokawa Shoten,1993). Examples of PEG-modifying reagent include ε-amino group modifierof lysine (Laid-Open Patent Publication Number S61-178926), carboxylgroup modifier of aspartic acid and glutamic acid (Laid-Open PatentPublication Number S56-23587), guanidino group modifier of arginine(Laid-Open Patent Publication Number H2-117920), and the like.

The antibody which has bonded to the protein can be obtained as a fusionantibody. In other words, the cDNA which encodes the antibody or afunctional fragment of the antibody is linked with the cDNA whichencodes a specific protein, and DNA which encodes the fused protein ofthe specific protein and antibody is constructed. This DNA is insertedinto an expression vector for a prokaryotic organism or eukaryoticorganism. This expression vector can be introduced and expressed in theprokaryotic organism or eukaryotic organism in order to produce thefused antibody which is bonded with the specific protein.

With regard to the antibody against FGF23 of the present invention orthe functional fragment of the antibody, by taking measurements throughimmunological methods such as ELISA (Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Chapter 14, 1988; Monoclonal Antibodies:Principles and Practice, Academic Press Limited, 1996) or measuring thebinding dissociation constant by biosensor Biacore (Journal ofImmunological Methods, 145: 229-240, 1991), and measuring the inhibitionactivity (Nature, 444: 770-774, 2006) of the promoter activity of theEarly growth response gene-1 by human FGF23 stimulation using klothoexpression cells, human FGF23 binding activity and the activity ofinhibiting the function of human FGF23 can be evaluated.

In the present invention, “human antibody” is defined as an antibodywhich is an expression product of an antibody gene derived from humans.Human antibody, as will be described later, can be obtained byintroducing the human antibody gene locus and by administering antigento transgenic animals having the ability to produce human antibody.Examples of these transgenic animals include mice. The method ofcreation of mice which can produce human antibody is described, forexample, in International Publication Number WO02/43478 pamphlet.

For the antibody of the present invention, examples include an antibody(C10 antibody) produced by C10 hybridoma as will be described inExamples later. C10 hybridoma has had an international deposition basedon the Budapest treaty with accession No. FERM ABP-10772 (a display foridentification: C10) at the Patent Organism Depository Center (Central6, 1-1 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Feb. 2, 2007at the independent administrative institution of the Advanced IndustrialScience and Technology.

The antibody or functional fragment of the present invention alsoincluded monoclonal antibodies or functional fragments thereofcomprising the heavy chain and/or light chain consisting of amino acidsequences with 1 or several amino acid deletions, substitutions, andadditions in each of the amino acid sequences for the heavy chain and/orlight chain which constructs the antibody or functional fragment. Here,of the “1 or several”, “several” is 9 or less, preferably 5 or less, andmore preferably 3 or less. Having 2 is especially preferred. A partialmodification (deletion, substitution, insertion, addition) of the aminoacid as described previously can be introduced into the amino acidsequence of the antibody of the present invention or functional fragmentby partially modifying the nucleotide sequence which encodes the aminoacid sequence. Partial modification of this nucleotide sequence can beintroduced using the conventional method of known site specificmutagenesis [Proc Natl Acad Sci USA., 81: 5662-5666, 1984]. The antibodyof the present invention includes antibodies of all immunoglobulinclasses and isotypes.

The antibody against FGF23 of the present invention can be produced bythe following production method. For example, FGF23 or a portion ofFGF23 or a conjugate of a portion of FGF23 and a suitable carriersubstance for increasing antigenicity (for example, bovine serum albuminand the like) are immunized together with an adjuvant (Freund's completeor incomplete adjuvant and the like) as necessary into non-human mammalssuch as human antibody producing transgenic mice. For FGF23, naturalFGF23 or recombinant FGF23 can be used. Alternatively, immunesensitization can be conducted by introducing the gene encoding FGF23into an expression vector and expressing the FGF23 protein inside theanimal. The monoclonal antibody is obtained by culturing the hybridomaobtained by fusing antibody producing cells obtained from immunesensitized animals and myeloma cells which do not have antibodyproduction ability, and by selecting the clones which produce themonoclonal antibodies showing specific affinity for the antigen used forimmunization.

The antibody of the present invention includes those that have beenconverted to a different subclass by genetic engineering modificationthat is known to those skilled in the art (for example, see EuropeanPatent Application EP314161). In other words, using DNA which encodesthe variable region of the antibody of the present invention, anantibody which is of a subclass that is different from the originalsubclass can be obtained using genetic engineering methods.

2. Producing the Antibody of the Present Invention

Producing a monoclonal antibody includes the following steps. In otherwords, (1) the antigen protein which is to be used as the immunogen orthe antigen protein expression vector is prepared, (2) afterimmunization by injecting the antigen inside the animal or by expressingthe antigen inside the animal, blood is sampled and its antibody titeris assayed, and after determining the time for spleen isolation,antibody production cells are prepared, (3) Myeloma cells are prepared,(4) the antibody production cell and myeloma are fused, (5) thehybridoma group which produces the target antibody is selected, (6) thehibridomas are divided into a single cell clone (cloning), (7)optionally, the hybridoma is cultured to produce large amounts ofmonoclonal antibody, or animals in which the hybridoma has beentransplanted are raised, and (8) the biological activity and therecognition specificity of the monoclonal antibody produced in this wayis assayed, or the property as a labeling reagent are assayed.

Hereinafter, the production method for the anti-FGF23 monoclonalantibody is described in detail following the above process. However,the production method for this antibody is not limited to this method.For example, antibody producing cells and myeloma from other than spleencells can also be used.

(1) Purification of the Antibody

Using genetic recombination technique, the DNA sequence which encodesFGF23 is integrated into a suitable expression plasmid. After FGF23 isproduced in a host such as E. coli or an animal cell or the like, thepurified FGF23 protein can be used. Because the primary structure ofhuman FGF23 protein is known [GenBank accession No. AAG09917, SEQ ID NO:4], a partial peptide from the amino acid sequence of FGF23 ischemically synthesized by methods known to those skilled in the art, andthis can also be used as the antigen.

(2) Preparation Step of Antibody Producing Cell

The antigen obtained as mentioned in (1) is mixed with a adjuvant suchas complete or incomplete Freund's adjuvant or aluminum potassium, andthe mixture is immunized into experimental animals as an immunogen. Forthe experimental animals, transgenic mice having the ability to producehuman derived antibodies are most suitably used. This type of mice isdescribed by the reference by Tomizuka et al [Tomizuka. et al., ProcNatl Acad Sci USA., 97: 722-727, 2000].

The immunogen administration method when immunizing mice can be any ofsubcutaneous injection, intraperitoneal injection, intravenousinjection, intradermal injection, intramuscular injection, foot padinjection, and the like. Intraperitoneal injection, foot pad injectionor intravenous injection is preferred.

The immunization can be conducted once or repeated several times at asuitable interval. Afterwards, the antibody titer against the antigen inthe serum of the immunized animal is measured. When animals with asufficiently high antibody titer are used as a supply source forantibody producing cells, the efficiency of later operations isincreased. In general, it is preferable to use antibody producing cellsderived from animals 3-5 days after the final immunization for thefollowing cell fusion.

Examples of the method used here for measuring antibody titer includeknown techniques such as radioimmunoassay (hereinafter referred to as“RIA method”), enzyme linked immunoabsorbent assay (hereinafter referredto as “ELISA method”), fluorescent antibody method, passivehemagglutination method, and the like. From the standpoint of detectionsensitivity, rapidity, accuracy, and the possibility of automatedoperation, RIA method or ELISA method is suitable.

According to the ELISA method for example, the measurement of antibodytiter of the present invention can be conducted by the followingprocedure. First, antigen against human antibody is absorbed onto thesolid phase surface of an ELISA 96 well plate for example. Furthermore,the solid phase surface which has not absorbed antigen is covered with aprotein unrelated to the antigen, such as bovine serum albumin (BSA).After rinsing the surface, it is allowed to contact with a seriallydiluted reagent as a primary antibody (for example, serum fromtransgenic mice having the ability to produce human antibodies) to makethe antigen described above which binds to the anti-FGF23 antibody inthe sample. Furthermore, an enzyme-labeled antibody against humanantibody is added as the secondary antibody to be allowed to bind to thehuman antibody. After washing, a substrate for the enzyme is added.Then, change in the light absorption caused by the color resulted fromthe substrate breakdown is measured to calculate the antibody titer.

(3) Preparation Step for Myeloma

For the myeloma, cells which do not have antibody production ability bythemselves and which are derived from mammals such as mouse, rat, guineapig, hamster, rabbit, or humans, and the like can be used. In general,cell lines obtained from mice, for example 8-azaguanine resistant mice(BALB/c derived) myeloma line P3X63Ag8U.1 (P3-U1) [Yelton, D. E. et al.,Current Topics in Microbiology and Immunology, 81: 1-7, 1978],P3/NSI/1-Ag4-1 (NS-1) [Kohler, G. et al., European J. Immunology, 6:511-519, 1976], Sp2/O-Ag14 (SP2/O) [Shulman, M. et al., Nature, 276:269-270, 1978], P3X63Ag8.653 (653) [Kearney, J. F. et al., J.Immunology, 123: 1548-1550, 1979], P3X63Ag8 (X63) [Horibata, K. andHarris, A. W., Nature, 256: 495-497, 1975] and the like are preferablyused. These cell lines are subcultured in a suitable medium, for example8-azaguanine medium [an RPMI-1640 medium supplemented which glutamine,2-mercaptoethanol, gentamycin, and fetal calf serum (FCS) as well as8-azaguanine], Iscove's Modified Dulbecco's Medium (IMDM), or Dulbecco'sModified Eagle Medium (DMEM). However, 3-4 days prior to cell fusion,the cell lines are subcultuted in a normal medium (for example DMEMmedium containing 10% FCS), and on the day of fusion, a cell number of2×107 or greater is prepared.

(4) Cell Fusion

The antibody producing cells are plasma cells and lymphocytes which aretheir precursor cells. These can be obtained from any site from theindividuals. In general, the spleen, lymph node, bone marrow, tonsils,peripheral blood, or any of these can be combined. In general, spleniccells are used most often.

After the final immunization, the site where the antibody producingcells is present, for example the spleen, is removed from mice whichhave achieved a prescribed antibody titer, and the splenic cells whichare the antibody producing cells are prepared. Next, splenic cells andmyeloma are fused. For the means for fusing the splenic cell and themyeloma obtained in step (3), the method that is used most generally isa method using polyethylene glycol. This method has relatively low celltoxicity and the fusion operation is also easy. This method has thefollowing procedure, for example.

The splenic cell and myeloma is washed well with serum-free medium (forexample DMEM) or a phosphate buffered saline (PBS). The splenic cell andmyeloma are mixed at a cell number ratio of around 5:1-10:1 and arecentrifuged. The supernatant is removed, and after loosening theprecipitated cell group, 1 mL of a serum-free medium containing 50%polyethylene glycol (molecular weight 1000-4000) (w/v) is instilled intothe cells while stirring. Afterwards, 10 mL of serum-free medium isslowly added, and afterwards, this is centrifuged. The supernatant isagain discarded, and the precipitated cells is suspended in a suitableamount of normal medium (referred to as HAT medium) which containssuitable amount of hypoxanthine/aminopterine/thymidine (HAT) solutionand human interleukin-6 (IL-6). The cells are aliquoted onto each wellof a culturing plate (henceforth referred to as “plate”), and culturedfor approximately 2 weeks at 37 degrees C. under 5% carbon dioxide gas.During this time, HAT medium is supplemented as needed.

(5) Selection of Hybridoma Group

When the myeloma cells described above is a 8-azaguanine resistantstrain, in other words, if it is ahypoxanthine/guanine/phosphoribosyltransferase (HGPRT) deficient strain,the myeloma cells which were not fused and fused cells of only myelomacells will not survive in HAT containing medium. On the other hand,fused cells of only antibody producing cells and hybridomas of antibodyproducing cell and myeloma cell can survive, but for the fused cells ofonly antibody producing cells have a limited lifespan. Therefore, bycontinuing to culture in a HAT-containing medium, only the hybridomaswhich are fused cells between antibody producing cells and myeloma cellswill survive. As a result, hybridomas can be selected.

For the hybridoma which is growing in colonies, medium exchange to amedium in which aminopterin is removed from HAT medium (henceforthreferred to as HT medium) is conducted. Afterwards, a portion of themedium supernatant is collected, and the anti-FGF23 antibody titer ismeasured by the ELISA method, for example.

Above, we showed an example of a method using an 8-azaguanine resistantcell line, but other cell lines can also be used according to theselection method for hybridomas. In these cases, the medium compositionto be used also changes.

(6) Cloning Step

By measuring the antibody titer with the same method as the antibodytiter measuring method as in (2), the hybridoma which has beendetermined to produce the specific antibody is transferred to anotherplate, and cloning is conducted. Examples of cloning methods include thelimiting dilution method in which the hybridoma are diluted so thatthere is one hybridoma contained per 1 well of a plate and this iscultured; soft agar method in which the hybridomas are cultured in asoft agar medium and the colonies are collected; a method in which onecell at a time is removed with a micromanipulator and this is cultured;“sorter cloning” in which a single cell is separated by a cell sorter,and the like. The limiting dilution method is simple and is often used.

With regard to the wells in which antibody titer has been seen, forexample, cloning is repeated 2-4 times by the limiting dilution method,and cells having a stable antibody titer, these are selected asanti-FGF23 monoclonal antibody producing hybridoma lines.

(7) Preparation of Monoclonal Antibody by Hybridoma Culturing

The hybridomas in which cloning has been completed are cultured byexchanging the medium from HT medium to normal medium. For large-scaleculturing, there are rotation culturing using a large-scale culturebottle, spinner culturing, or culturing using a hollow fiber system, andthe like. By purifying the supernatant in large-scale culturing using amethod known to those skilled in the art such as gel filtration and thelike, anti-FGF23 monoclonal antibody can be obtained. In addition, bygrowing this hybridoma intraperitoneally in the same strain of mouse(for example BALB/c) or nu/nu mouse, rat, guinea pig, hamster, or rabbitor the like, peritoneal fluid containing large amounts of anti-FGF23monoclonal antibody can be obtained. A simple method for purificationuses commercial monoclonal antibody purification kits (for example,MAbTrap GII kit; GE Healthcare Bioscience Co.) and the like.

The monoclonal antibodies obtained in this way have high antigenspecificity against FGF23.

In addition, recombinant antibody can be prepared by cloning the genewhich encodes human monoclonal antibody from the antibody producingcells of the hybridoma and the like, incorporating the gene into asuitable vector and introducing into a host (for example, mammalian cellline, E. coli, yeast cell, insect cell, plant cell, and the like), andusing genetic recombination technology (Delves, P. J., ANTIBODYPRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY, Shepherd, P. and Dean C.,Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS, Goding, J. W.,Monoclonal Antibodies: principles and practice., 1993 ACADEMIC PRESS).

The present invention includes the nucleic acids which contain thegenetic sequence for the antibody of the hybridoma which produces theantibody of the present invention, in particular the nucleic acid forthe heavy chain variable region and light chain variable region of theantibody produced by the hybridoma of the present invention that will bedescribed later. Here, nucleic acid includes DNA and RNA. Furthermore,the present invention includes the nucleic acid of the mature portion inwhich the region encoding the signal sequence from the nucleic acid ofthe heavy chain variable region and light chain variable region of thepresent invention has been removed. Furthermore, in addition to thenucleic acids described above, the nucleic acid of the present inventionincludes the nucleic acids having the codons corresponding to the aminoacids of the amino acid sequence of the antibody of the presentinvention and to the amino acids of the antibody heavy chain variableregion and/or light chain variable region of this antibody.

In order to prepare the gene which encodes the monoclonal antibody fromthe hybridoma, a method is used in which DNA encoding each of the Lchain V region, L chain C region, H chain V region and H chain C regionof the monoclonal antibody is prepared by PCR method or the like. Forthis, oligoDNA designed from the anti-FGF23 antibody gene or the aminoacid sequence is used as the primer. For the template, DNA prepared fromthe hybridoma can be used. These DNAs are incorporated into one suitablevector and this is introduced into a host and is expressed, or elsethese DNAs are each incorporated into a suitable vector, andco-expressed.

For the vector, phages or plasmids which can grow autonomously in thehost microorganisms are used. For the plasmid DNA, examples includeplasmids from E. coli, Bacillus subtilis, or yeast, and the like. Forthe phage DNA, examples include λ phage.

The host used in transformation is not limited as long as it is onewhich can express the target gene. Examples include bacteria (E. coli,Bacillus subtilis, and the like), yeast, animal cells (COS cells, CHOcells and the like), and insect cells and the like.

Methods for introducing genes into a host are known, and there are manyexamples of methods (for example, a method which uses calcium ion,electroporation method, spheroplast method, lithium acetate method,calcium phosphate method, lipofection method, and the like). Inaddition, examples of methods for introducing the gene into animalswhich will be described later include microinjection method, method ofintroducing genes into ES cells using electroporation method andlipofection method, nuclear transplantation, and the like.

In the present invention, the transformant is cultured, and theanti-FGF23 antibody is obtained by collecting from the culture product.Here, “culture product” signifies any of (a) culture supernatant, (b)cultured cells or cultured bacteria or their homogenate, (c) secretionsof the transformant. In order to culture the transformant, a mediumsuitable for the host is used, and stationary culture method, culturemethod by roller bottle and the like are used.

After culturing, when the target antibody is produced inside thebacteria or inside the cell, the antibody is collected by homogenizingthe bacteria or cell. In addition, when the target antibody is producedoutside the bacteria or outside the cell, the culture solution can beused directly, alternatively the bacteria or cells are removed bycentrifugation or the like. Afterwards, the target antibody can beisolated and purified from the culture product by general biochemicalmethods using, singly or in combination, various chromatographies usedfor isolation and purification of proteins.

Furthermore, using transgenic animal creation techniques, animal hostsin which the gene of the target antibody is incorporated into endogenousgenes, for example transgenic cattle, transgenic goat, transgenic sheep,or transgenic pig are created. A large amount of monoclonal antibodyderived from the antibody gene can be obtained from the milk secretedfrom these transgenic animals (Wright, G., et al., Bio/Technology 9:830-834, 1991). When culturing the hybridoma in vitro, the hybridoma isgrown, maintained and stored according to the various conditions of theproperties of the cultured cell the experimental research and culturemethods and the like. Known nutrition medium or various nutrition mediumderived and prepared from known basic medium can be used to produce themonoclonal antibody in the culture supernatant.

(8) Assay of the Monoclonal Antibody

Determining the isotype and subclass of the monoclonal antibody obtainedin this manner can be conducted in the following manner. First, examplesof the identification method include Ouchterlony method, ELISA method,or RIA method, and the like. The Ouchterlony method is simple, but whenthe concentration of the monoclonal antibody is low, a concentratingprocedure is necessary. On the other hand, when ELISA method or RIAmethod is used, the culture supernatant is reacted directly with theantigen absorbed solid phase, and as a secondary antibody, antibodiesresponding to various immunoglobulin isotypes, and subclasses can beused to identify the isotype and subclass for the monoclonal antibody.

Furthermore, the quantification of the protein can be conducted byFolin/Lowry method and by a method which calculates light absorption at280 nm [1.4 (OD280)=immunoglobulin 1 mg/mL].

Identification of recognition epitopes of the monoclonal antibody(epitope mapping) is conducted as follows. First, the partial structuresof various molecules that monoclonal antibodies recognize are created.For the creation of partial structures, there is a method in which knownoligopeptide synthesis techniques are used to create various partialpeptides of the molecule, and a method in which, using geneticrecombination techniques, the DNA sequence which encodes the targetpartial peptide is incorporated in a suitable expression plasmid, andthe peptides are produced inside or outside of the host such as E. colior the like. However, in general, both methods are combined for theabove objective. For example, a series of polypeptides in which theantigen protein has been sequentially shortened at random lengths fromthe carboxy terminal or amino terminal is created using geneticrecombination techniques known to those skilled in the art. Afterwards,the reactivity of the monoclonal antibody to these polypeptides isstudied, and recognition sites are roughly determined.

Afterwards, for further detail, the oligopeptide of the correspondingportion, or variants and the like of these peptides are synthesized byoligopeptide synthesis techniques known to those skilled in the art. Inorder to define the epitopes, the binding of the monoclonal antibodiescontained as an active ingredient in the agent for prevention ortreatment of the present invention to these peptides is studied,alternatively the competitive inhibition activity of the peptides to thebinding of the monoclonal antibodies to the antigen is studied. As asimple method for obtaining various oligopeptides, commercial kits (forexample, SPOTs kit (Genosis Biotechnologies), a series of multipinpeptide synthesis kits which uses multipin synthesis method (Chiron Co,)and the like) can be used.

(9) Producing the Antibody Fragment

The antibody fragment is produced by genetic engineering methods orproteochemical methods based on the antibody described in (7) of theabove.

For the genetic engineering method, the gene which encodes the targetantibody fragment is constructed and expressed using a suitable hostsuch as animal cell, plant cell, insect cell, E. coli and the like, andthe antibody fragment is purified.

For the proteochemical method, proteases such as pepsin, papain, and thelike are used for site specific cleavage, and purification is conducted.

For the antibody fragment, examples include peptides comprising Fab, F(ab′)2, Fab′, scFv, diabody, dsFv, CDR and the like. The productionmethod for each of the antibody fragments is described in detail below.

(i) Production of Fab

Proteochemically, Fab can be created by treating IgG with proteasepapain. After treatment with papain, if the original antibody is an IgGsubclass having protein A binding ability, by passing through a proteinA column, IgG molecules and Fc fragments are separated, and a uniformFab can be recovered (Monoclonal Antibodies: Principles and Practice,third edition, 1995). If the antibody is an IgG subclass with no proteinA binding ability, with ion exchange chromatography, Fab is recoveredfrom the fraction which is eluted at low salt concentrations (MonoclonalAntibodies: Principles and Practice, third edition, 1995). In addition,for genetic engineering of Fab, E. coli is used in most cases, or insectcells and animal cells and the like are used to produce Fab. Forexample, DNA which encodes the V region of the antibody described in 2(7) above is cloned into a Fab expression vector to construct a Fabexpression vector. For the Fab expression vector, anything can be usedas long as DNA for Fab can be incorporated and expressed. An example ispIT106 (Science, 240: 1041-1043, 1988) and the like. The Fab expressionvector is introduced into a suitable E. coli, and Fab can be generatedand stored in an inclusion body or periplasma layer. From the inclusionbody, the Fab can be activated by a refolding method normally used withproteins. In addition, when expression is in the periplasma layer,active Fab is discharged into the culture supernatant. After refoldingor from the culture supernatant, by using a column with bound antigen, auniform Fab can be purified (Antibody Engineering, A Practical Guide, W.H. Freeman and Company, 1992).

(ii) Production of F (ab′)2

Proteochemically, F (ab′)2 is produced by treating IgG with proteasepepsin. After treating with pepsin, a uniform F (ab′)2 is recoveredthrough the same purification operation as with Fab (MonoclonalAntibodies: Principles and Practice, third edition, Academic Press,1995). In addition, it can be created by a method in which Fab′described in the following (iii) is treated with a maleimide such aso-PDM or bis maleimide hexane and the like, and thioether bonds areformed or it can be created by a method in which it is treated with DTNB[5,5′-dithiobis(2-nitrobenzoic acid)], and S—S bonds are formed(Antibody Engineering, A Practical Approach, IRL PRESS, 1996).

(iii) Production of Fab′

Fab′ can be obtained by treating F (ab′)₂ described in the above (ii)with a reducing agent such as dithiothreitol, and the like. In addition,with genetic engineering, Fab′ can be created by using E. coli in mostcases or insect cells or animal cells and the like. For example, DNAwhich encodes the V region of the antibody described in the above 2 (7)is cloned into a Fab′ expression vector and a Fab′ expression vector canbe constructed. For the Fab′ expression vector, anything can be used aslong as DNA for Fab′ can be incorporated and expressed. An example ispAK19 (BIO/TECHNOLOGY, 10: 163-167, 1992) and the like. Fab′ expressionvector is introduced into a suitable E. coli. Fab′ can be generated andaccumulated in an inclusion body or in the periplasma layer. From theinclusion body, Fab′ is activated by the refolding method used normallyin proteins. In addition, when expressed in the periplasma layer,bacteria is homogenized by treatment with partial digestion by lisozyme,osmotic shock, sonication, and the like, and this can be recovered fromoutside the bacteria. After refolding or from the bacterial homogenate,a uniform Fab′ can be purified by using a protein G column and the like(Antibody Engineering, A Practical Approach, IRL PRESS, 1996).

(iv) Production of scFv

By genetic engineering, scFv can be produced by using a phage or E. colior insect cells or animal cells and the like. For example, DNA whichencodes the V region of the antibody described in 2 (7) can be clonedinto a scFv expression vector to construct a scFv expression vector. Forthe scFv expression vector, anything can be used as long as DNA for scFvcan be incorporated and expressed. Examples include pCANTAB5E (GEHealthcare Bioscience Co.), pHFA (Human Antibodies & Hybridomas, 5:48-56, 1994) and the like. scFv expression vector is introduced into asuitable E. coli. By infecting with a helper phage, a phage in whichscFv is expressed on the phage surface as fused with a phage surfaceprotein can be obtained. In addition, scFv can be generated andaccumulated in the inclusion body or in the periplasma layer of the E.coli in which the scFv expression vector has been introduced. From theinclusion body, activated scFv can be obtained by the refolding methodnormally used for proteins. In addition, when expressed in theperiplasma layer, bacteria are homogenized by treatment with partialdigestion by lisozyme, osmotic shock, sonication, and the like, and thisis recovered from outside the bacteria. After refolding or from thebacterial homogenate, a uniform scFv can be purified by using positiveion exchange chromatography and the like (Antibody Engineering, APractical Approach, IRL PRESS, 1996).

(v) Production of Diabody

By genetic engineering, diabody can be produced mainly using E. coli aswell as insect cells and animal cells. For example, DNA is produced inwhich VH and VL of the antibody described in above 2 (7) are linked sothat the amino acid residues encoded by the linker are 8 residues orless and cloned in a diabody expression vector to construct theexpression vector for diabody. Any vector can be used as a diabodyexpression vector as long as it can be integrated with diabody DNA andexpress diabody DNA. Examples include pCANTAB5E (GE HealthcareBioscience), pHFA (Human Antibodies Hybridomas, 5, 48, 1994) and thelike, diabody can be generated and accumulated in the inclusion body orin the periplasma layer of the E. coli in which the diabody expressionvector has been introduced. From the inclusion body, activated diabodycan be obtained by the refolding method normally used for proteins. Inaddition, when expressed in the periplasma layer, bacteria arehomogenized by treatment with partial digestion by lisozyme, osmoticshock, sonication, and the like, and this is recovered from outside thebacteria. After refolding or from the bacterial homogenate, a uniformdiabody can be purified by using positive ion exchange chromatographyand the like (Antibody Engineering, A Practical Approach, IRL PRESS,1996).

(vi) Production of dsFv

dsFv can be created mainly using E. coli as well as insect cells andanimal cells by genetic engineering. First, mutations are introduced atappropriate sites of DNA which encodes VH and VL of antibody describedin (ii), (iv) and (v), and DNA in which the coded amino acid residuesare replaced with cysteine is produced. Each DNA produced can be clonedin dsFv expression vector to construct expression vectors for VH and VL.Any vector can be used as an dsFv expression vector as long as it can beintegrated with and express dsFv DNA. For example, pUL19 (ProteinEngineering, 7: 697-704, 1994) and the like can be used. The expressionvector for VH and VL can be introduced into an appropriate E. coli andgenerated products can be accumulated in inclusion body or periplasmalayer. VH and VL are obtained from inclusion body and periplasma layer,mixed and converted to dsFv with activity by the refolding method whichis employed in normal protein processing. After refolding, furtherpurification by ion-exchange chromatography and gel-filtration can becarried out (Protein Engineering, 7: 697-704, 1994).

(vii) Production of CDR Peptide

Peptides containing CDR can be produced by the chemical synthesis methodsuch as Fmoc method or tBoc method and the like. Also, CDR peptideexpression vector can be produced by producing DNA which encodes apeptide containing CDR and by cloning the DNA produced in an appropriateexpression vector. Any vector can be used as an expression vector aslong as it can be integrated with and express DNA that encodes CDRpeptide. For example, pLEX (Invitrogen) and pAX4a+ (Invitrogen) may beused. The expression vector can be introduced into an appropriate E.coli and generated products can be accumulated in inclusion body orperiplasma layer. CDR peptide is obtained from inclusion body orperiplasma layer and can be purified by ion exchange chromatography andgel-filtration (Protein Engineering, 7: 697-704, 1994).

3. Characteristic of the Antibody of the Present Invention and theFunctional Fragment Thereof.

The antibody of the present invention and the functional fragmentthereof possesses any of the characteristic below.

(a) FGF23 binding test; binds to the full length protein having aminoacid residues from 25th to 251st of SEQ ID NO: 4 of FGF protein.

(b) In vitro test; inhibits the action of FGF23 in an assay, by whichthe action of FGF23 can be detected. An example of the method fordetecting the action of FGF23 in vitro is the activation of the promoterof the early growth response gene-1 by human FGF23 stimulation (Nature,444: 770-774, 2006).

(c) In vivo test; inhibits the activity of endogenous FGF23 andincreases serum phosphorous concentration and serum 1,25D concentrationwhen administered to human. The extent of the increase of the serumphosphorous concentration and serum 1,25D concentration is greatercompared to conventional antibody, 2C3B antibody (the mouse monoclonalantibody against FGF23 protein disclosed in WO03/057733, anti-FGF23antibody produced by hybridoma of Accession No. FERM BP-7838) and alsothe duration of increased level of serum phosphorous concentration andserum 1,25D concentration is long. For example, the duration of elevatedserum phosphorous concentration is about 3 times or longer, preferablyabout 5 times as that of 2C3B antibody, and the duration of elevatedserum 1,25D concentration is about 1.5 times or longer, preferably about2.5 times as that of 2C3B antibody when administered to cynomolgusmonkey.

The present invention also includes a nucleic acid which encodes anamino acid sequence of the antibody to FGF23 of the present invention.The nucleic acid may be DNA or RNA. The nucleic acid of the presentinvention is, preferably, a nucleic acid which encodes an amino acidsequence of antibody produced by hybridoma C10. An example is a nucleicacid encoding the amino acid sequence of the heavy chain variable region(heavy chain nucleotide sequence of C10 antibody), which is coded by thenucleotide sequence from at position 58 C to at position 408 A shown inSEQ ID NO: 11. In addition, another example is a nucleic acid encodingthe amino acid sequence of the light chain variable region, which iscoded by the nucleotide sequence from G at position 67 to A at position384 shown in SEQ ID NO: 13.

II. Pharmaceutical Compositions

A formulation which is a pharmaceutical composition comprising the humananti-FGF23 antibody of the present invention or the functional fragmentthereof is included in the scope of the present invention. Such aformulation, preferably, includes in addition to the antibody and thefunctional fragment thereof, a physiologically acceptable diluents orcarriers and may be a mixture with other drugs such as other antibody orantibiotics. Appropriate carriers include physiological saline,phosphate buffered saline, phosphate buffered saline glucose solution,and buffered physiological saline, but not limited to these. Further,the antibody may be freeze-dried and may be re-constituted by addingabove buffer solution when needed, and then used. Administration routesinclude oral administration, or parenteral administration such asintraoral, tracheobronchial, endorectal, subcutaneous, intramuscular andintravenous administration, and preferred administration route isintravenous administration. Administration can be conducted in variousformulations and the formulations include, aerosol, capsules, tablets,granules, syrup, emulsion, suppositories, injections, ointments andtapes.

Liquid preparations such as emulsion and syrup can be produced usingadditives for example: water; saccharides such as sucrose, sorbitol andfructose; glycols such as polyethylene glycol, propylene glycol; oilssuch as sesame oil, olive oil and soy bean oil; preservatives such asp-hydroxybenzoate esters; flavors such as strawberry flavor andpeppermint.

Capsules, tablets, powder and granules can be produced using additivesfor example: excipients such as lactose, glucose, sucrose and mannitol;disintegrators such as starch and sodium alginate; lubricants such asmagnesium stearate and talc; binders such as polyvinyl alcohol,hydroxypropyl cellulose and gelatin; surface active agents such as fattyacid ester; plasticizers such as glycerin.

In the injections, additives can be used include; water; saccharidessuch as sucrose, sorbitol, xylose, trehalose, fructose and the like;sugar alcohols such as mannitol, xylitol and sorbitol; buffers such asphosphate buffer, citrate buffer and glutamate buffer; surface activeagents such as fatty acid ester.

An appropriate formulation for parenteral administration includesinjections, suppositories, aerosol and the like. In case of injections,it is normally provided in the form of unit dosage ampules or multipledosage containers. It may be powder which is re-dissolved, when in use,in an appropriate carrier, for example pyrogen-free sterile water. Theseformulations contain additives such as emulsifier, suspending agent andthe like, which are generally used for formulating in thesecompositions. Methods for injection include, for example intravenousinfusion, intravenous injection, intramuscular injection,intraperitoneal injection, subcutaneous injection, intradermal injectionand the like. Also, the dosage is different according to the age of theadministration subject, administration route, frequency ofadministration, and can be changed widely.

A suppository is prepared using a carrier such as cacao butter,hydrogenated fat or carboxylic acid. Aerosol can be prepared using theantibody of the present invention of the functional fragment thereofitself, or using a carrier which does not irritate oral and respiratorytract mucosa of a recipient (patient) and can disperse theaforementioned antibody and the functional fragment thereof as fineparticles to facilitate absorption.

In particular, examples of a carrier include lactose, glycerin and thelike. Depending on the characteristic of the aforementioned antibody orthe functional fragment thereof and the characteristic of the carrier tobe used, formulation such as aerosol, dry powder and the like can bechosen. Also, the components shown as examples of additives for oralformulation can be added to these parenteral formulations.

The dosage may vary according to symptoms, age, body weight but normallyin oral administration, about 0.01 mg-1000 mg per day for an adult isadministered. This can be administered once or divided into severalbatches. In parenteral administration, about 0.01 mg-1000 mg can beadministered by subcutaneous, intramuscular or intravenous injection peradministration.

The present invention includes the antibody of the present invention, orthe functional fragment thereof, or a preventive or therapeutic methodfor diseases described below using a pharmaceutical compositioncontaining thereof, and furthermore, the present invention include a useof the antibody of the present invention or the functional fragmentthereof for manufacturing an agent for preventive or therapeutic of thediseases described below.

Diseases that can be prevented or treated by the antibody of the presentinvention or the functional fragment thereof include diseases havingexcessive activity of FGF23 such as tumor-induced osteomalachia, ADHR,XLH, fibrous dysplasia, McCune-Albright syndrome, and a diseaseaccompanying abnormal mineral metabolism such as autosomal recessivehypophosphataemia. Further, improving effects can be expected forsyndromes associated with these diseases such as, hypophosphataemia,bone mineralization failure, bone pain, muscle weakness, skeletaldeformity, growth disorder, low blood 1,25D and the like. Since FGF23plays an important role under the physiological condition, the calciummetabolism control activity of FGF23, which is mediated by the controlof phosphorous metabolism and vitamin D metabolism, can be regulated bythe antibody of the present invention and the functional fragmentthereof, and thus, they can be used preventively and therapeutically fordiseases caused by abnormality in mineral metabolism and vitamin Dmetabolism, such as osteoporosis, rickets (including hypophosphatemicrickets and vitamin D-resistant rickets), hypercalcaemia, hypocalcaemia,ectopic calcification, osteosclerosis, Paget's disease,hyperparathyroidism, hypoparathyroidism, pruritus and the like. Further,the antibody of the present invention and the functional fragmentthereof can also be used preventively or therapeutically for diseasescaused by the complication of kidney failure and dialysis for kidneyfailure, represented by renal osteodystrophy, dialysis osteopathy, renaltubular dysfunction. On the other hand, 1,25D has been reported to haveactivities not only on mineral metabolism such as calcium metabolism asdescribed above but also cell growth inhibitory effect, celldifferentiation promotion activity and the like. Thus the antibody ofthe present invention and the functional fragment thereof can be usedtherapeutically and preventively against diseases caused by the cellswhose growth and differentiation are regulated by 1,25D.

Also, it is known that, in tumor-induced osteomalachia, overproductionof FGF23 by the tumor causes pathology. Therefore it may be conceivablethat retraction of the tumor may be induced by using the antibody of thepresent invention linked with a radioactive substance such asradioactive isotope and the like, or with therapeutic reagent of varioustoxins such as low molecular weight drugs and by accumulating thepresent antibody in the FGF23 overproducing tumor.

III. Formulation Example

The formulation containing the antibody of the present invention or thefunctional fragment thereof, is provided as an ampule of sterilesolution dissolved in water or pharmacologically acceptable solution orsuspension. Also, a sterile powder formulation (it is preferable tofreeze dry the molecule of the present invention) may be placed in anampule and may be diluted in use with a pharmacologically acceptablesolution.

EXAMPLES

Following is the detailed description of the present invention byExamples, but it does not mean that the present invention is limited tothese descriptions of Examples only.

Example 1 Preparation of an Expression Vector for Recombinant HumanFGF23

(1) Construction of an Expression Vector for Human FGF23H Protein

cDNA encoding human FGF23 was amplified by using the human cDNA libraryof the responsible tumor for tumor-induced osteomalachia as a template,a F1EcoRI primer (SEQ ID NO: 1) and a LH is Not primer (SEQ ID NO: 2)and LA-Taq DNA polymerase and by conducting 35 cycles of a PCR stepconsisting of heating at 96° C. for 1 min, then at 96° C. for 30 sec, at55° C. for 30 sec and at 72° C. for 30 sec. The F1EcoRI primer wasannealed to a sequence present at further upstream of the 5′ side of thenucleotide sequence encoding human FGF23 and adds an EcoRI restrictionsite at the 5′ side of the nucleotide sequence encoding human FGF23 inthe amplified fragment. The LH is Not primer comprises a sequence whichanneals to the sequence at the 5′ side of the stop codon of thenucleotide sequence encoding human FGF23, a sequence encoding theterminal codon which follows the sequence encoding the His6-tag sequence(His-His-His-His-His-His) and a NotI restriction site. As a result, theamplified fragment encodes human FGF 23 protein in which the His6-tagsequence is added at the carboxy terminal and has a NotI restrictionsite at the downstream thereof. This amplified fragment was digestedwith EcoRI and Not I, and ligated to an animal cell expression vector,pcDNA3.1Zeo (Invitrogen) which was similarly digested with EcoRI andNotI. The expression vector constructed in such a way was cloned and thenucleotide sequence was determined to confirm that the expression vectorencodes the target, human FGF23 protein to which the His6-tag sequencewas added. This vector is called pcDNA/hFGF23H.

F1EcoRI: (SEQ ID NO: 1) CCGGAATTCAGCCACTCAGAGCAGGGCACG LHisNot:(SEQ ID NO: 2) ATAAGAATGCGGCCGCTCAATGGTGATGGTGATGATGGATGAACTTGGC GAA(2) Construction of an Expression Vector for Human FGF23 Protein

A fragment was amplified by using pcDNA/hFGF23H as a template, theF1EcoRI primer and a LNot primer (SEQ ID NO: 3) and LA-Taq DNApolymerase and by conducting 25 cycles of a PCR step consisting ofheating at 94° C. for 1 min, then at 94° C. for 30 sec, at 55° C. for 30sec and at 72° C. for 1 min. After terminating the reaction, thefragment encoding human FGF23 was digested with EcoRI and NotI, and thenpurified. This was cloned by inserting at the EcoRI and NotI restrictionsites of pEAK8/IRES/EGFP vector, an animal cell expression vector, pEAKS(Edge Biosystem), to which the intramolecular ribosomal entry sequence(IRES) and enhanced green fluorescent protein (EGFP) were ligated. Thenucleotide sequence of thus obtained plasmid was determined to confirmthat it encodes human FGF23 protein. This vector was calledpEAK8/IRES/EGFP/hFGF23.

LNot: (SEQ ID NO: 3) ATAAGAATGCGGCCGCTCAGATGAACTTGGCGAA

Example 2 Expression of Recombinant Human FGF23 and Recombinant MutantHuman FGF23H Protein

(1) pcDNA/hFGF23H was Linearized by Cleaving the FspI Restriction Site

pcDNA/hFGF23H was linearized by cleaving the FspI restriction site inthe ampicillin resistant gene in the vector and purified, and then mixedwith CHO Ras clone-1 cells (Shirahata, S., et al., Biosci BiotechBiochem, 59: 345-347, 1995) and transfected to the cells byelectroporation using Gene Pulser II (Bio Rad). After culturing thesecells in MEM α medium (Gibco BRL) containing 10% FCS for 24 h, Zecocin(Invitrogen) was added to a final concentration of 0.5 mg/ml and thenthe cells were cultured for a week. Cells attached and grown werereleased by trypsinization and cloned by the limited dilution method inthe presence of Zecocin at the final concentration of 0.3 mg/ml toobtain a multiplicity of cloned cells. The cell expressing human FGF23Hmost efficiently was identified by the Western blotting method. Culturesupernatants of each cloned cell were collected and were subjected toSDS-polyacrylamide gel electrophoresis, and then proteins weretransferred to a PVDF membrane (Millipore). A signal derived fromFGF-23H protein was detected at about 32 kDa by using anti-His-tag(carboxy terminal) antibody (Invitrogen) and ECL photo-luminescentsystem (GE Healthcare Bioscience).

As the result, the highest expression was found in a clone called #20,which was named as CHO-OST311H and deposited at International PatentOrganism Depositary (IPOD) National Institute of Advanced IndustrialScience and Technology (AIST) Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan on Aug. 11, 2000 (Deposition No.: FERM BP-7273). In thepresent description, CHO-OST311H is called CHO-hFGF23H.

(2) Obtaining of Human FGF23 Expressing Cells

Transfection of pEAK8/IRES/EGFP/hFGF23 vector to CHO Ras clone-1 cellswas carried out by the gene transfection method using a membrane fusionlipid. CHO Ras clone-1 cells were cultured in 6 well plates until about60% of the bottom of the well was covered by the cells, and then theculture medium was removed and 1 ml of MEMα medium without serum wasadded. Each of 2.5 μg of the vector to be introduced and 10 μl ofTransfectam (Registered Trademark)(Promega) was mixed with 50 μl of MEMαmedium without serum, and then both solutions were mixed and leftstanding for 10 min. The mixtures were added to wells of 6 well platesprepared beforehand. After incubating for 2 hours, the culture mediumcontaining DNA was removed, replaced with a medium containing 10% FCS,and the culture was incubated overnight. Next day, Puromycin (Sigma) wasadded to a final concentration of 5 μg/ml to select drug resistantcells. The drug resistant cells thus obtained were cloned by the limiteddilution method. Further, the cell line expressing the target proteinmost efficiently was obtained by Western blotting method. This cell linewas called CHO-hFGF23.

(3) Expression and Detection of Recombinant Human FGF23 Protein inAnimal Cells

Western blotting of the recombinant in the culture supernatant ofCHO-hFGF23H using the antibodies against the carboxy terminal anti-His6tag sequence detected bands of about 32 kDa and about 10 kDa. These 2bands were excised out of the gel, and the amino acid sequences of theamino terminals were determined. In the larger molecular weight band(about 32 kDa), the sequence from amino acid 25 of SEQ ID NO: 4 wasdetected and it appeared to be human FGF23 protein from which the signalsequence was removed during the process of excretion. On the other hand,in the band having a smaller molecular weight, the sequence from aminoacid 180 of SEQ ID NO: 4 was confirmed and it turned out that thisfragment was the carboxy terminal fragment produced by the cleavagebetween amino acid 179 and 180. Also, the presence of a polypeptidehaving the sequence from amino acid 179 to the amino terminal (aminoterminal fragment) was recognized by detecting it using polyclonalantibody which recognizes the amino terminal side of human FGF23(International Publication No. WO02/14504 Pamphlet).

Similarly, in the culture supernatant of CHO-hFGF23 having no His 6-tagsequence, the cleavage between amino acid residue 179 and 180 wasconfirmed (International Publication No. WO02/14504 Pamphlet).Therefore, following operations were carried out to separate and purifythe full length human FGF23 protein, which appears not to be cut, havingthe sequence from amino acid 25 to amino acid 251 of SEQ ID NO: 4(sometimes referred to as full length FGF23) from the amino terminal orcarboxy terminal fragments.

(4) Purification of Recombinant Full Length Human FGF23 Protein

The culture supernatant of CHO-hFGF23 was filtered through SuperCap(Registered Trade Mark) (Pall Gelman Laboratory) which is a membranefilter having 0.2 μm pore size, and the filtrate was passed throughSP-Sepharose FF (GE Healthcare Bioscience). Substances having weakaffinity to the column was washed and eluted with 50 mM sodium phosphatebuffer, pH 6.7. This fraction contained the carboxy terminal fragmentgenerated by the cleavage between amino acid 179 and 180. Protein heldby the column was eluted with NaCl concentration gradient from 0 to 0.7M, and full length human FGF23 protein was observed in the fractioneluted with about 0.3 M NaCl. Next, full length human FGF23 protein wasabsorbed to Talon Superflow (Registered Trade Mark) (Clonetech), whichis a metal affinity column, washed with 50 mM sodium phosphate buffer,pH 6.7 and then eluted by adding imidazole at different concentrations.The fraction containing the target protein was absorbed to a SPsepharose FF column and eluted for further purification.

Human FGF23 amino acid sequence (SEQ ID NO: 4)MLGARLRLWV CALCSVCSMS VLRAYPNASP LLGSSWGGLIHLYTATARNS YHLQIHKNGH VDGAPHQTIY SALMIRSEDAGFVVITGVMS RRYLCMDFRG NIFGSHYFDP ENCRFQHQTLENGYDVYHSP QYHFLVSLGR AKRAFLPGMN PPPYSQFLSRRNEIPLIHFN TPIPRRHTRS AEDDSERDPL NVLKPRARMTPAPASCSQEL PSAEDNSPMA SDPLGVVRGG RVNTHAGGTG PEGCRPFAKF I

Example 3 Production of Mice Producing Human Antibody (KM Mice)

Mice producing complete human antibody for preparation of humanmonoclonal antibody have the homozygous genetic background fordestructed endogenous both Ig heavy chain and kappa-light chain and alsofor having the chromosome 14 fragment (SC20) containing the human Igheavy chain gene loci and the human Ig kappa chain trans gene (KCo5) atthe same time. These mice were produced by cross-breeding the strain Amouse which has the human Ig heavy chain gene loci and the strain Bmouse which has the human Ig kappa chain trans gene. The strain A ishomozygous for destructed endogenous both Ig heavy chain and the kappalight chain, and is a mouse line having the chromosome 14 fragment(SC20) which can be transmitted to offsprings. This line of mouse isdescribed, for example, in the report by Tomizuka et al., (Tomizuka, etal., Proc Natl. Acad. Sci. USA., 97: 722-727, 2000). Also, the strain Bis homozygous for destructed endogenous both Ig heavy chain and thekappa light chain and is a transgenic mouse line having the human Igkappa chain trans gene (KCo5). This line of mouse is described, forexample, in the report by Fishwild et al., (Nat. Biotechnol., 14;845-851, 1996).

In the following experiments, used are individual mice, which areobtained by crossing a male strain A mouse and a female strain B mouse,or a male strain B mouse and a female strain A mouse, and in which humanIg heavy chain and kappa light chain are detected at the same time inthe serum [Ishida & Lonberg, IBC's 11th Antibody Engineering, Abstract2000]. Furthermore, the mice producing human antibody can be obtainedfrom Kirin Beer Company by contracting.

Example 4 Preparation of Human Monoclonal Antibody Against Human FGF23

(1) Obtaining a Hybridoma Producing Human Monoclonal Antibody AgainstHuman FGF23

Monoclonal antibodies used in the present Examples are preparedaccording to the general method described in, such as, “Introduction tomonoclonal antibody experimental manipulation” by Tamio Ando et al.,Published by Kodansha, 1991. Full length human FGF23 protein prepared inExample 2 was used as an immunogen, and the human antibody producingmice produced in Example 3 which produce human immunoglobulin wereimmunized.

First, to prepare human monoclonal antibody against FGF23, purified fulllength human FGF23 protein prepared in Example 2 was mixed with RIBIadjuvant (Corixa) and inoculated intraperitoneally to human antibodyproducing mice at a dose of 20 μg/mouse as the first immunization.Similar to the first immunization, the mixture of purified FGF23 andRIBI adjuvant was inoculated total 3 times at 2 weeks intervals. Fivemice were used for immunization, blood samples were collected after thethird immunization, and the presence of human IgG antibody against FGF23in sera was confirmed by the enzyme labeled immunosorbent assay (ELISA)method as described below. The mouse was selected which showed thehighest serum value by the ELISA using FGF23 fixed on the solid phasewith anti-FGF23 protein mouse monoclonal antibody, 3CIE, which wasdisclosed in International Publication No. WO03/057733 Pamphlet (antiFGF23 antibody produced by the hybridoma deposited as FERM BP-7839) andwas immunized by 20 μg of full length human FGF23 protein/mouse via tailvein administration 3 days before taking the spleen out as describedbelow.

The spleen was surgically taken out of the immunized mice, immersed in10 ml of the DMEM containing 350 mg/mL of sodium bicarbonate, 50units/mL of penicillin, 50 μg/mL streptomycin and no serum (Invitrogen,called DMEM without serum, hereinafter) and crushed on a mesh (Cellstrainer: Falcon)) using a spatula. The cell suspension which passedthrough the mesh was centrifuged to precipitate cells, and then thecells were washed twice with DMEM without serum and suspended in DMEMwithout serum to measure the cell number. While, myeloma cells, SP2/0(ATCC No. CRL-1581) were cultured in DMEM (Invitrogen) containing 10%FCS (Sigma) (called DMEM with serum, hereinafter) at 37° C. under 5%carbon dioxide gas so that cell density does not exceed 1×106 cells/mL.These myeloma cells were similarly washed with DMEM without serum,suspended in the same medium and counted. Recovered spleen cellsuspension and mouse myeloma cell suspension were mixed at the cellnumber ratio 5:1, centrifuged and the supernatant was completelyremoved. To this cell pellet, 1 mL of 50% (w/v) polyethylene glycol 1500(Boehringer-Manheim) as a fusion agent was added slowly while stirringthe pellet with a tip of a pipette, and then 1 mL of DMEM without serumthat was pre-warmed at 37° C. was added slowly in 2 portions and further7 mL of DMEM without serum was added. After centrifuging, thesupernatant was removed and the fused cells thus obtained were subjectedto screening by the limited dilution method as described below. Thehybridoma selection was carried out by culturing in DMEM containing 10%FCS and IL-6 (10 ng/mL) (or 10% hybridoma cloning factor (called HCF,hereinafter): Biobase), and hypoxanthine (H), aminopterin (A) andthymidine (T) (called HAT, hereinafter: Sigma). Further, single cloneswere obtained by the limited dilution method using DMEM containing HT(Sigma), 10% FCS and 10% HCF. Culturing was conducted in 96 wellmicrotiter plates (Becton, Dickinson). Selection of hybridoma clonesproducing anti-FGF23 human monoclonal antibody (screening) andcharacterization of human monoclonal antibody produced by respectivehybridomas were conducted by the enzyme labeled immunosorbent assay(ELISA) as described below. As the results, many hybridomas wereobtained which contained human immunoglobulin γ chain (hIgγ) and humanimmunoglobulin light chain κ, and produced human monoclonal antibodyhaving the specific reactivity to human FGF23. Among a number ofhybridomas obtained, 2 clones (C10 and C15) were particularly obtainedas hybridomas producing an antibody which recognizes the FGF23 protein.Furthermore, in all the Examples described below including this Example,the hybridoma clones that produce the anti-FGF23 human monoclonalantibody of the present invention were designated using symbols. Stillfurther, “antibody” affixed before or after these symbols indicates theantibody produced by the hybridoma or recombinant antibody produced byhost cells carrying the antibody gene (full length or variable region)isolated from the hybridoma. Also, to the extent where the contextclearly indicates, the name of the hybridoma clone may indicate the nameof the antibody. The hybridoma clone C10 has been deposited atInternational Patent Organism Depositary (IPOD) National Institute ofAdvanced Industrial Science and Technology (AIST) Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, Japan on Feb. 2, 2007 (Deposition No.:FERM ABP-10772) (Label for ID: C10).

(2) Purification of C 10 and C 15 Antibody from the Culture Supernatantof the Hybridoma

C10 and C15 hybridoma obtained in Example 4 was conditioned to eRDFmedium (Kyokuto Seiyaku) containing bovine insulin (5 μg/ml,Invitrogen), human transferrin (5 μg/ml, Invitrogen), ethanoamine (0.01mM, Sigma), sodium selenite (2.5×10-5 mM, Sigma), 1% Low IgG FetalBovine Serum (Hyclone). The hybridoma was cultured in a flask and theculture supernatant was recovered. The culture supernatant wasaffinity-purified using Protein G Fast Flow gel (GE Healthcare,Bioscience), PBS(−) as an absorption buffer and 0.1 M glycine buffer (pH2.8) as an elution buffer. The eluted fraction was adjusted to about pH7.2 by adding 1 M Tris (pH 9.0). The antibody solution thus prepared wasreplaced by PBS using a Sephadex G25 desalting column (NAP column; GEHealthcare Bioscience) and sterilized by filtration with a membranefilter MILLEX-GV with 0.22 μm pore size (Millipore) to obtain purifiedC10 and C15 antibody. The concentration of the purified antibody wascalculated by measuring 280 nm absorption and by assuming 1 mg/mL as 1.4OD.

Example 5

Obtaining the Antibody Gene Encoding C10 Antibody and Determination ofthe Sequence Thereof

(1) Synthesis of cDNA of C10 Antibody

To obtain the DNA fragment containing the variable regions of humanantibody heavy chain and light chain which are expressed in C10hybridoma, cloning by the 5′ RACE method (5′ rapid amplification of cDNAends) was carried out using primers specific to the constant regions ofheavy and light chain of human antibody. More particularly, the cloningwas carried out using the BD SMART RACE cDNA Amplification Kit (BectonDickinson Bioscience Clonetech) following the manufacturer'sinstruction.

RNA extraction reagent, ISOGEN (Nippon Gene), was added to C10 hybridomaand 15 μg of total RNA was purified as the material for cDNA synthesisaccording to the manufacturer's instruction. The 1st strand of cDNA wasprepared using about 1 μg of each purified total RNA as a template. Allthe reagents and enzymes except RNA used were provided by the BD SMARTRACE cDNA Amplification Kit.

In the 1st strand cDNA synthesis,

Total RNA 1 μg/3 μl 5′CDS 1 μl SMART Oligo 1 μlthe reaction mixture with the above composition was incubated at 70° C.fir 2 min, and then

5 × Buffer 2 μl DTT 1 μl dNTP mix 1 μl PowerScript Reverse Transcriptase1 μlwere added and incubated at 42° C. fir 1.5 h.

Further, 50 μl of Tricine-EDTA Buffer was added and then incubated at72° C. for 7 min to obtain the 1st strand of cDNA.

(2) Amplification of the Heavy Chain Gene and the Light Chain Gene byPCR and Confirmation of the Nucleotide Sequences.

(2)-1; Amplification of the Heavy Chain Gene and the Light Chain Gene byPCR.

To amplify the cDNA of the gene encoding C10 antibody, followingreaction mixture was prepared and subjected to PCR, using a PCR primerset of the 3′ primer having the sequence specific to human antibody (theparticular sequence is described later) and the 5′ primer (Universalprimer A mix) that hybridizes specifically to the sequence added to the5′ terminal of the cDNA synthesized by the BD SMART RACE cDNAAmplification Kit, and KOD-Plus-DNA polymerase (Toyobo) as PCR enzyme.

sterile H2O 28 μl  1st strand cDNA 2.5 μl   KOD-Plus-buffer (10×) 5 μldNTP Mix (2 mM) 5 μl MgSO4 (25 mM) 2 μl KOD-Plus-(1 unit/μl) 1 μlUniversal primer A mix (UPM) (10×) 5 μl Gene specific primers (GSP) (10μM) 1.5 μl   Total volume 50 μl 

For the amplification of the heavy chain gene, the set of UPM primer inthe SMART RACE cDNA Amplification Kit and IgG 1p primer (SEQ ID NO: 5)was used, while for the amplification of the light chain gene, the setof UPM primer and hk-2 (SEQ ID NO: 6) primer was used.

IgG1p: TCTTGTCCACCTTGGTGTTGCTGGGCTTGTG (SEQ ID NO: 5) hk-2:GTTGAAGCTCTTTGTGACGGGCGAGC (SEQ ID NO: 6)

Also, the reaction condition used is as follows.

-   -   5 cycles of 94° C./30 sec and 72° C./3 min were repeated,    -   5 cycles of 94° C./30 sec, 70° C./30 sec and 72° C./3 min were        repeated, and    -   25 cycles of 94° C./30 sec, 68° C./30 sec and 72° C./3 min were        repeated.

Further, this reaction mixture 2 μl was diluted by adding 98 μl ofTricine-EDTA Buffer, and the second (nested) PCR was carried out using 5μl of the diluted mixture as a template. The composition of the PCRreaction solution is as follows:

sterile H2O 30 μl  The first PCR reaction solution (50 fold dilution) 5μl KOD-Plus-buffer (10X) 5 μl dNTP Mix (2 mM) 5 μl MgSO4 (25 mM) 2 μlKOD-Plus-(1 unit/μl) 1 μl Nested Universal primer A (NUP; 10 μM) 1 μlGene specific primers (GSP) (10 μM) 1 μl Total volume 50 μl 

As a primer set for the amplification of the heavy chain gene in theabove reaction, NUP primer (in the SMART RACE cDNA Amplification Kit;Becton Dickinson Bioscience Clonetech) and hh2 primer (SEQ ID NO: 7)were used, and for the amplification of the light chain gene, UPM primerand hk-5 primer (SEQ ID NO: 8) were used. The reaction temperaturecondition was as follows: at 94° C. as the initial temperature for 1min, then 20 cycles of 94° C./5 sec, 68° C./10 sec and 72° C./3 min wererepeated. Finally heating at 72° C./7 min was carried out.

hh2: GCTGGAGGGCACGGTCACCACGC (SEQ ID NO: 7) hk-5:AGGCACACAACAGAGGCAGTTCCAGATTTC (SEQ ID NO: 8)

(2)-2; Determination of the Nucleotide Sequence of the Antigen Gene

The amplified heavy chain PCR fragment (hereinafter, referred to asHV[C]: consisting of the 5′-untranslated region-leader sequence,variable region (HV) and a part of constant region ([C]) of the Hchain), and the amplified light chain PCR fragment (hereinafter,referred to as LV[C]: consisting of the 5′-untranslated region-leadersequence, variable region (LV) and a part of constant region ([C]) ofthe L chain) were recovered by ethanol precipitation, and then subjectedto agarose gel electrophoresis. Recovered fragments were purified by aDNA purification kit using a membrane, QIAquick Gel Extraction Kit(Qiagen). The purified HV[C] amplified fragment or LV[C] amplifiedfragment was subcloned in PCR 4 Blunt-TOPO vector of Zero Blunt TOPO PCRCloning Kit (Invitrogen) and the nucleotide sequence of the insert DNAwas analyzed for the plasmid DNA of the clone obtained. The primers usedfor DNA nucleotide sequence were M13-20FW (SEQ ID NO: 9) and M13RV (SEQID NO: 10).

M13-20FW: GTAAAACGAC GGCCAGTG (SEQ ID NO: 9) M13RV: CAGGAAACAGCTATGAC(SEQ ID NO: 10)

DNA nucleotide sequence encoding the heavy chain variable region andlight chain variable region, and amino acid sequence of heavy chainvariable region and light chain variable region of C10 antibody are showbelow.

<C10 heavy chain nucleotide sequence> (from the ATG initiation codon tothe DNA sequence encoding the carboxy terminal amino acid residues ofthe variable region) (SEQ ID NO: 11)

        10         20         30         40         50         60ATGGACTGGA CCTGGAGGGT CTTCTGCTTG CTGGCTGTAG CTCCAGGTGC TCACTCCCAG        70         80         90        100        110        120GTGCAGCTGG TGCAGTCTGG GGCTGAGGTG AAGAAGCCTG GGGCCTCAGT GAAGGTTTCC       130        140        150        160        170        180TGCAAGGCAT CTGGATACAC CTTCACCAAC CACTATATGC ACTGGGTGCG ACAGGCCCCT       190        200        210        220        230        240GGACAAGGGC TTGAGTGGAT GGGAATAATC AACCCTATTA GTGGTAGCAC AAGTAACGCA       250        260        270        280        290        300CAGAAGTTCC AGGGCAGAGT CACCATGACC AGGGACACGT CCACGAGCAC AGTCTACATG       310        320        330        340        350        360GAGCTGAGCA GCCTGAGATC TGAGGACACG GCCGTGTATT ATTGTGCGAG AGATATTGTG       370        380        390        400        408GATGCTTTTG ATTTCTGGGG CCAAGGGACA ATGGTCACCG TCTCTTCA

<C10 heavy chain amino acid sequence> (to the leader sequence andvariable region) (SEQ ID NO: 12) (Underlined amino acid residuesrepresent the leader sequence as a secretion signal)

        10         20         30         40         50         60MDWTWRVFCL LAVAPGAHSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN HYMHWVRQAP        70         80         90        100        110        120GQGLEWMGII NPISGSTSNA QKFQGRVTMT RDTSTSTVYM ELSSLRSEDT AVYYCARDIV       130      136 DAFDFWGQGT MVTVSS

<C10 light chain nucleotide sequence> (from the ATG initiation codon tothe DNA sequence encoding the carboxy terminal amino acid residues ofthe variable region) (SEQ ID NO: 13)

        10         20         30         40         50         60ATGGACATGA GGGTCCCCGC TCAGCTCCTG GGGCTTCTGC TGCTCTGGCT CCCAGGTGCC        70         80         90        100        110        120AGATGTGCCA TCCAGTTGAC CCAGTCTCCA TCCTCCCTGT CTGCATCTGT AGGAGACAGA       130        140        150        160        170        180GTCACCATCA CTTGCCGGGC AAGTCAGGGC ATTAGCAGTG CTTTAGTCTG GTATCAGCAG       190        200        210        220        230        240AAACCAGGGA AAGCTCCTAA GCTCCTGATC TATGATGCCT CCAGTTTGGA AAGTGGGGTC       250        260        270        280        290        300CCATCAAGGT TCAGCGGCAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGCAGCCTG       310        320        330        340        350        360CAGCCTGAAG ATTTTGCAAC TTATTACTGT CAACAGTTTA ATGATTACTT CACTTTCGGC       370        380    384 CCTGGGACCA AAGTGGATAT CAAA

<C10 light chain amino acid sequence> (to the leader sequence andvariable region) (SEQ ID NO: 14) (Underlined amino acid residuesrepresent the leader sequence as a secretion signal)

        10         20         30         40         50         60MDMRVPAQLL GLLLLWLPGA RCAIQLTQSP SSLSASVGDR VTITCRASQG ISSALVWYQQ        70         80         90        100        110        120KPGKAPKLLI YDASSLESGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQFNDYFTFG       128 PGTKVDIK

Further, in the gene sequence of C10 antibody subcloned in PCR 4Blunt-TOPO vector, a part of the constant region of the human antibodysequence was cloned and the DNA nucleotide sequence of this region wasalso analyzed. The result indicated that the presence of the sequenceencoding the amino acid residue 118 to 191 in the heavy chain constantregion which is shown by the EU index by Kabat et al., was confirmed andwas in complete agreement with the amino acid sequence of human IgG1,and thus it was determined that the subclass of C10 antibody was IgG1.In addition, the antibody gene encoding C15 antibody was obtained andthe sequence thereof was determined by using the same method.

Example 6 Construction of Recombinant C10 Antibody Expression Vector

Production of C10 Expression Vector (Process Scheme is Shown in FIG. 1)

The DNA of LV (light chain leader sequence+variable region) of C10antibody was amplified by PCR by KOD-Plus-DNA polymerase using obtainedplasmid DNA containing LV[C] chain of C10 antibody as a template andprimers C10_L5_Bg1 (SEQ ID NO: 15) and C10_L3_Bsi (SEQ ID NO: 16) whichwere designed to add restriction enzyme sites (5′ terminal BglII, 3′terminal BsiWI) for linkage to the ends. The reaction temperaturecondition was: after heating for 1 min at the starting temperature 94°C., a cycle of 94° C./5 sec and 68° C./45 sec was repeated 35 times anda final heating 72° C./7 min. The amplified DNA fragment was digestedwith restriction enzymes BglII and BsiWI and purified by recovering 400by DNA from agarose gel electrophoresis. While the vector DNA, N5KG1-ValLark vector (IDEC Pharmaceuticals, a modified vector of N5KG1 (U.S. Pat.No. 6,001,358)) was similarly digested with restriction enzymes BglIIand BsiWI sequentially, subjected to dephosphorylation treatment withAlkaline Phosphatase (E. coli C75) (Takara Shuzo Co., Ltd.) and thenrecovered as a little smaller than about 9 kb DNA after purification byagarose gel electrophoresis and DNA purification kit. These 2 fragmentswere ligated with T4 DNA ligase and transfected to E. coli DH10B toobtain transformants. Plasmid DNA of the transformants, which containedthe insert DNA, was subjected to DNA nucleotide sequence analysis, andplasmid DNA, N5KG1_C10_Lv, in which LV of C10 antibody was inserted inframe at 5′ upstream of human antibody light chain constant region ofN5KG1-Val Lark was obtained. Next, the HV (the leader sequence+variableregion of heavy chain) of C10 antibody was inserted to the plasmidvector in which LV was inserted (N5KG1_C10-_Lv). The HV was amplified byPCR using the plasmid DNA containing the HV[C] of C10 antibody subclonedin pCR4Blunt-TOPO vector as a template and the primers, C10_H5_Sal (SEQID NO: 17) and C10_H3_Nhe (SEQ ID NO: 18) designed to add restrictionenzyme sites (SalI at the 5′ terminal, NheI at 3′ terminal) for linkageto the ends. The reaction temperature condition was: after heating for 1min at the starting temperature 94° C., a cycle of 94° C./5 sec and 68°C./45 sec was repeated 35 times and a final heating 72° C./7 min.Purified HV amplified DNA fragment was subcloned in pCR4Blunt-TOPOvector, and the insert DNA of thus obtained clones of plasmid DNAanalyzed by sequencing. The primers used for DNA sequencing wereM13-20FW and M13RV described above. The inserted part of the subcloneswas analyzed by DNA sequencing, and the plasmid DNA (TOPO_C10_Hv), whichhad no difference with the template HV and the primer parts were alsothe same sequence as designed, was selected. This DNA was digested withrestriction enzymes, SalI and NheI, subjected to agarose gelelectrophoresis, and the DNA fragment which was about 420 bp wasrecovered and purified, and was ligated using T4 DNA ligase toN5KG1_C10_Lv DNA (about 9 kb) which was similarly subjected torestriction enzyme treatment (SalI and NheI) and dephosphorylation. Theligation product was introduced into E. coli DH10B and the targetplasmid DNA was selected from the transformants thus obtained. Theantibody expressing plasmid DNA, N5KG1_C10_IH (clone #1) obtained inthis way was mass produced and purified, and it was confirmed that nochange was introduced during the cloning process in the DNA nucleotidesequence of the entire region of L chain and H chain and around theinserted site (FIGS. 2 and 3). Confirmation of the DNA sequence wascarried out by using primers of SEQ ID NO: 19-25. Simplified map of C10antibody expression vector is shown in FIG. 4. In addition, arecombinant C15 antibody expression vector was constructed by using thesame method.

C10_L5_Bg1: (SEQ ID NO: 15) GAGAGAGAGATCTCTCACCATGGACATGAGGGTCCCCGCTC10_L3_Bsi: (SEQ ID NO: 16) AGAGAGAGAGCGTACGTTTGATATCCACTTTGGTCCCAGGGCC10_H5_Sal: (SEQ ID NO: 17) AGAGAGAGAGGTCGACCACCATGGACTGGACCTGGAGGGTCTTCC10_H3_Nhe: (SEQ ID NO: 18) AGAGAGAGAGGCTAGCTGAAGAGACGGTGACCATTGTCCChh-4: (SEQ ID NO: 19) GGTGCCAGGGGGAAGACCGATGG hh-1: (SEQ ID NO: 20)CCAAGGGCCCATCGGTCTTCCCCCTGGCAC CMVH903F: (SEQ ID NO: 21)GACACCCTCATGATCTCCCGGACC CMVHR1303: (SEQ ID NO: 22)TGTTCTCCGGCTGCCCATTGCTCT SEQU4618: (SEQ ID NO: 23)TCTATATAAGCAGAGCTGGGTACGTCC hk-1: (SEQ ID NO: 24)TGGCTGCACCATCTGTCTTCATCTTC SEQU1783: (SEQ ID NO: 25)GGTACGTGAACCGTCAGATCGCCTGGA

Example 7 Preparation of Recombinant C10 Antibody

C10 antibody expressing cells were produced by introducing theconstructed C10 antibody expression vector to host cells. A strain ofdihydrofolate reductase (DHFR) deletion mutant CHO DG44 cells(hereinafter, referred to as CHO cells, IDEC Pharmaceuticals),conditioned to a serum-free medium, EX-CELL325 PF medium (JRH,containing 2 mM glutamine, 100 units/ml penicillin, 100 μg/mlstreptomycin, hypoxanthine and thymidine (HT) supplement (1:100)(Invitrogen)) was used as host cells for expression. Introduction of thevector to the host cells was carried out by electroporation. The genewas introduced to 4×106 CHO cells by electroporation by linearizingabout 2 μg of the C10 expression vector with a restriction enzyme AscIand using a BioRad Electroporator at 350 V, 500 μF, and then cells wereseeded to 96 cell culture plates. After introducing the vector to thecells, G418 was added and the culture was continued. After confirmingcolonies, strains expressing antibody were selected. The selected CHOcell lines were cultured in EX-CELL-325 PF medium (containing 2 mMglutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, hypoxanthineand thymidine (HT) supplement (1:100) (Invitrogen)) under 5% CO2. Theculture supernatant was absorbed to a Mabselect Protein A column (GEHealthcare Bioscience), washed with PBS and eluted with 20 mM Na-citrateand 50 mM NaCl (pH 3.4) buffer. The eluate was neutralized to pH 7.0with 50 mM sodium phosphate pH 7.0. The conductivity was adjusted to 4.0ms/cm or below by diluting about 1.5 fold with deionized water. Next,the sample was applied to a linked column of Q-Sepharose (Hitrap Q HP,GE Healthcare Bioscience) and SP-Sepharose (Hitrap SP FF, GE HealthcareBioscience) for absorption, washed with 20 mM sodium phosphate buffer(pH 5.0) and then eluted with PBS(−). The antibody solution thusprepared was filter sterilized through a 0.22 μm pore size membranefilter, MILLEX-GV (Millipore). The concentration of purified C10antibody was calculated by measuring 280 nm absorption and by assuming 1mg/mL as 1.4 OD. In addition, a recombinant C15 antibody was prepared byusing the same method.

Example 8 Construction of Cynomolgus Monkey FGF23 Protein ExpressionVector

(1) Construction of Cynomolgus Monkey FGF23 Protein Expression Vector

To EDTA treated venous blood of cynomolgus monkey, 5% Dextran T-2000 (GEHealthcare Bioscience) suspended in PBS (−) was mixed at the ratio of2:1 to precipitate red blood cells. Then, the supernatant was layered ontop of a lymphocyte separation solution (Ficoll-Plaque) (GE HealthcareBioscience) and centrifuged to obtain the lymphocyte fraction.Lymphocytes thus obtained were suspended in ISOGEN-LS (Nippon Gene), andtotal lymphocyte RNA of cynomolgus monkey was obtained according to theattached protocol. From this total lymphocyte RNA of cynomolgus monkey,the lymphocyte cDNA library of cynomolgus monkey was prepared usingFirst Strand cDNA Synthesis Kit (Invitrogen) according to the attachedprotocol. cDNA encoding cynomolgus monkey FGF23 was amplified using thelymphocyte cDNA library of cynomolgus monkey as a template, monkeyFGF23FW primer (SEQ ID NO: 26) and monkey FGF23RV primer (SEQ ID NO:27), and KOD plus DNA polymerase (Toyobo), and incubating at 94° C. for5 min, then carrying out 45 cycles of a PCR step of heating at 94° C.for 20 sec, at 55° C. for 30 sec and at 72° C. for 50 sec. The monkeyFGF23FW primer anneals to a sequence present in the 5′ upstream regionof the nucleotide sequence encoding human FGF23 and adds the EcoRIrestriction site to the 5′ side of the FGF23 coding region in theamplified fragment. The monkey FGF23RV primer contains a sequence whichanneals to the sequence containing the stop codon of the human FGF23coding region, and the Not I restriction site. This amplified fragmentwas digested with EcoRI and NotI, and cloned by inserting at the EcoRIand NotI restriction sites of pEAK8/IRES/EGFP vector, which is anexpression vector pEAK8 (Edge Biosystem) to which internal ribosomeentry site (IRES) and enhanced green fluorescent protein (EGFP) arelinked. The nucleotide sequence of thus obtained plasmid was determinedto confirm that it encodes a cynomolgus monkey FGF23 protein. Thisvector was called pEAK8/IRES/EGFP/monkeyFGF23. The nucleotide sequenceand amino acid sequence of cynomolgus monkey FGF23 obtained in thepresent Example are shown in SEQ ID NO: 28 and 29, respectively.

(SEQ ID NO: 26) monkeyFGF23FW: GGAATTCCACCATGTTGGGGGCCCGCCTCAGGCT(SEQ ID NO: 27) monkeyFGF23RV: ATTTGCGGCCGCTAGATGAACTTGGCGAAGGGGC

Nucleotide sequence of cynomolgus monkey FGF23 (SEQ ID NO: 28)

ATGTTGGGGGCCCGCCTCAGGCTCTGGGTCTGTGCCTTGTGCAGCGTCTGCAGCATGAGCGTCATCAGAGCCTATCCCAATGCCTCCCCATTGCTCGGCTCCAGCTGGGGTGGCCTGATCCACCTGTACACAGCCACAGCCAGGAACAGCTACCACCTGCAGATCCACAAGAATGGCCACGTGGATGGCGCACCCCATCAGACCATCTACAGTGCCCTGATGATCAGATCAGAGGATGCTGGCTTTGTGGTGATTACAGGTGTGATGAGCAGAAGATACCTCTGCATGGATTTCGGAGGCAACATTTTTGGATCACACTATTTCAACCCGGAGAACTGCAGGTTCCGACACTGGACGCTGGAGAACGGCTACGACGTCTACCACTCTCCTCAGCATCACTTTCTGGTCAGTCTGGGCCGGGCGAAGAGGGCCTTCCTGCCAGGCATGAACCCACCCCCCTACTCCCAGTTCCTGTCCCGGAGGAACGAGATCCCCCTCATCCACTTCAACACCCCCAGACCACGGCGGCACACCCGGAGCGCCGAGGACGACTCGGAGCGGGACCCCCTGAACGTGCTGAAGCCCCGGGCCCGGATGACCCCGGCCCCGGCCTCCTGCTCACAGGAGCTCCCGAGCGCCGAGGACAACAGCCCGGTGGCCAGCGACCCGTTAGGGGTGGTCAGGGGCGGTCGGGTGAACACGCACGCTGGGGGAACGGGCCCGGAAGCCTGCCGCCCCTTCGCCAAGTTCATCTAG

Amino acid sequence of cynomolgus monkey FGF23 (SEQ ID NO: 29)

MLGARLRLWV CALCSVCSMS VIRAYPNASP LLGSSWGGLIHLYTATARNS YHLQIHKNGH VDGAPHQTIY SALMIRSEDAGFVVITGVMS RRYLCMDFGG NIFGSHYFNP ENCRFRHWTLENGYDVYHSP QHHFLVSLGR AKRAFLPGMN PPPYSQFLSRRNEIPLIHFN TPRPRRHTRS AEDDSERDPL NVLKPRARMTPAPASCSQEL PSAEDNSPVA SDPLGVVRGG RVNTHAGGTG PEACRPFAKF I(2) Preparation of Supernatant of Cynomolgus Monkey FGF23 ExpressingCells

pEAK8/IRES/EGFP/monkey FGF23 was transiently transfected to PEAK rapidcells (Edge Biosystem) by the calcium phosphate method, and theirculture supernatant was obtained.

Example 9 Investigation for Binding of C10 to Antibody Cynomolgus MonkeyFGF23

The fact that C10 antibody binds not only to human FGF23 but alsocynomolgus monkey FGF23 was investigated by the following method usingsandwich ELISA. C10 antibody prepared in Example 4, 2C3B antibody andhuman IgG1 control antibody were diluted in 50 mM NaHCO3 solution to aconcentration of 5 μg/ml and added to each well of 96 well microtiterplates for ELISA (Maxisorp (Registered Trade Name), Nunc), incubated at4° C. for 12 hours. Thus, C10 antibody, 2C3B antibody and human IgG1control antibody as a control were absorbed to microplates. Next, thesesolutions were removed, and a blocking reagent (SuperBlock (RegisteredTrade mark) Blocking buffer, PIERCE) was added to each well, incubatedat room temperature for 30 min and then each well was washed twice withTris-buffered saline (T-TBS) containing 0.1% Tween20. To each well ofthe microtiter plate to which anti-FGF23 antibodies were coated, fulllength human FGF23 protein purified in Example 2 or the expressing cellsupernatant of cells expressing cynomolgus monkey FGF23 prepared inExample 8 was added after diluting to appropriate concentrations,reacted to antibody in solid phase for 2 hours, and then each well waswashed twice with Tris-buffered saline (T-TBS) containing 0.1% Tween20.Next, biotin labeled 3C1E antibody at 3 μg/ml was added and incubated atroom temperature for 1.5 hours to bind biotin labeled 3C1E antibody tohuman or cynomolgus monkey FGF23 bound to the antibody in solid phase.After washing with T-TBS, horseradish peroxidase labeled streptavidin(DAKO) diluted 5000 fold was reacted for 1 hour and washed 3 times withT-TBS. Next, a substrate buffer containing tetramethylbenzidine (DAKO)was added to each well and incubated at room temperature for 30 min. Thereaction was stopped by addition of 0.5 M sulfuric acid to each well.Absorption at the wavelength of 450 nm with reference wavelength of 570nm was measured using a microplate reader (MTP-300, Colona ElectricCo.). Reactivity of human full length FGF23 protein and the culturesupernatant of cynomolgus monkey FGF23 expressing cells were compared bydiluting with factor of 3. The result is shown in FIGS. 5A and B. Asclearly shown in FIG. 5A, the reactivity of C10 antibody or 2C3Bantibody in solid phase to human full length FGF23 protein is about thesame. To serially diluted culture supernatant of cynomolgus monkey FGF23expressing cells under the conditions, not much difference is observedbetween the reactivity of C 10 antibody and 2C3B antibody (FIG. 5B).That is, C10 antibody, like 2C3B antibody, was proven to be able to bindto human and cynomolgus monkey FGF23.

Example 10 Comparison of the Effect of C10 Antibody and 2C3B Antibody onNormal Cynomolgus Monkey Blood Phosphorous Concentration and Blood 1α,25 Dihydroxy Vitamin D Concentration

FGF23 has activities of excreting phosphorous from the kidney, reducingserum phosphorous concentration as well as inhibiting vitamin Dactivating enzyme and reducing blood 1α, 25 dihydroxy vitamin D(hereinafter referred to as 1,25D) concentration (InternationalPublication No. WO02/14504 Pamphlet). It has been demonstrated thatadministration of antibody, such as 2C3B antibody and the like, whichhas a suppressive effect, that is, neutralizing activity, on FGF23, tonormal mice causes inhibition of endogenous FGF23 action and an increaseof serum phosphorous concentration and serum 1,25D concentration(International Publication No. WO03/057733 Pamphlet). Thus, it has beenstrongly suggested that antibody having neutralizing activity on FGF23has therapeutic effect on human diseases including tumor-inducedosteomalachia, XLH and the like which are caused by excessive FGF23.Therefore, C10 antibody obtained in the present invention wasinvestigated for FGF23 neutralizing activity in vivo. In particular,since its pharmacological effect on human is expected, the neutralizingeffect was measured in monkeys, which are evolutionally more closelyrelated to humans compared to species such as rodents, by using thesuppression of the function of endogenous FGF23, increase of serumphosphorous concentration and increase of serum 1,25D concentration asindexes. Experiments were conducted using a mouse antibody, 2C3Bantibody, as a comparative control for C10 antibody.

Effect of C10 antibody and 2C3B antibody on the increase of serumphosphorous concentration was compared in untreated normal cynomolgusmonkeys by the following method. C10 antibody produced in Example 4 wasused. Experimental animals used were female cynomolgus monkeys of 2-3years old and body weight 2-4 kg. 3 animals were used in each group ofthe solvent administration and 2C3B antibody administration, and 4animals were used in the C10 administration group. C10 and 2C3Bantibodies were prepared in PBS (−) at a concentration of 3 mg/ml andused as an administration solution. The solvent, PBS (−), was used as anegative control. C10 and 2C3B antibodies were administered once fromthe brachial cephalic vein at a flow rate of 1 ml/min and amount of 3mg/kg and 1 ml/kg. Serum phosphorous concentration was measured using Ltype Wako inorganic phosphorous reagent (Wako Pure Chemical Industries)and a Hitachi Clinical Analyzer Model 7180 (Hitachi, Ltd.). Serum 1,25Dconcentration was measured using 1, 25 (OH)2D RIA Kit [TFB](Immunodiagnostic System). Measurements were carried out at day 0.5, 1,2, 3, 5, 7, 10, 14, 21, 28, 35, 42, and 49 after the administration ofantibody. Data were shown in average+/−standard error. FIG. 6 shows thetransition of serum phosphorous concentration in periodically collectedblood samples up to 10 days after the administration of each antibody.In the PBS (−) administered group, the serum phosphorous concentrationwas almost constant during the test period, while in the C10 antibodyand 2C3B antibody administered groups a clear increase of the serumphosphorous concentration was observed when compared with before theadministration and PBS (−) administered group. The day when the highestserum phosphorous concentration was observed in both C10 antibodyadministered group and 2C3B antibody administered group was 5 days afterthe administration of the antibodies. At this time point, the serumphosphorous concentration in PBS(−) group, 2C3B antibody group and C10antibody group was 5.28 mg/dl, 8.10 mg/dl and 9.59 mg/dl, respectively.Comparing the serum phosphorous concentration of 2C3B antibody group andthe C10 antibody group at 5 days after the administration of antibodywith the serum phosphorous concentration of the PBS (−), the increase inthe 2C3B antibody group was 2.82 mg/dl, while that of C10 antibody groupwas 4.31 mg, suggesting that C10 antibody induced about 1.5 times orhigher increase in the serum phosphorous concentration compared to the2C3B antibody (FIG. 7). Thus the increase effect in the serumphosphorous concentration in C10 antibody administered group is markedlyhigher compared to that in the 2C3B antibody administered group.Further, at 10 days after the administration the serum phosphorousconcentration in the 2C3B antibody administered group was at the samelevel as that in PBS (−) group, while the serum phosphorousconcentration in the C10 administered group (8.76 mg/dl) was stillmaintaining higher level than the highest level (8.10 mg/dl) in the 2C3Bantibody administered group (FIG. 6). Further, the increased serumphosphorous concentration by C10 antibody is sustained far longer thanthat by 2C3B antibody. The duration, in which the significant differenceof the serum phosphate concentration from the PBS (−) group wasobserved, was 7 days for the 2C3B group, while it was surprisingly 35days, about 5 times longer, in the C10 antibody group. Similarly, for1,25D concentration, after the administration C10 antibody demonstrateda marked increase and elongation of the sustained increased durationcompared to 2C3B antibody (FIG. 8). These results demonstrate that incynomolgus monkeys C 10 antibody have more powerful increasing activityfor serum phosphorous concentration and serum 1,25D concentration, thatis, having more powerful FGF23 neutralizing activity. The currenttreatment for hypophosphatemic rickets in XLH at this time requires alarge dose of multiple administrations of phosphorous and vitamin Dformulations per day to barely maintain the normal range of thephosphorous concentration. There are reports of poor compliance ofpatients due to the plurality of administrations to take. The fact thatin the single administration of C10 antibody in the present study, asustained raising activity on serum phosphorous concentration and serum1,25D concentration was observed suggests that C10 antibody has possiblya marked advantage as a therapeutic drug for hypophosphatemia overconventional therapy.

Example 11 Confirmation of Reactivity of C15 Antibody to Human andCynomolgus Monkey FGF23

pEAK8/IRES/EGFP/hFGF23 prepared in Example 1 or pEAK8/IRES/EGFP/monkeyFGF23 prepared in Example 8 was transiently transfected into PEAK rapidcells (Edge Biosystem) by the calcium phosphate method. Each culturesupernatant was collected 3 days after introduction. Western blotting ofthe collected culture supernatant was performed using C15 antibodyprepared in Example 13 as a primary antibody (FIG. 9). As a result, C15was shown to bind to cynomolgus monkey FGF23, similarly to human FGF23.

Example 12 Comparison of the Effect of C10 Antibody and C15 Antibody onBlood Phosphorous Concentration and Blood 1α, 25 Dihydroxy Vitamin DConcentration in Normal Cynomolgus Monkeys

Example 11 demonstrated that C15 antibody has binding activity withhuman and cynomolgus monkey FGF23 recombinant proteins as does C10antibody. Subsequently, FGF23 neutralizing activity of C10 antibody andC15 antibody in vivo was compared by administering the antibodies tonormal cynomolgus monkeys. The neutralizing activity on cynomolgusmonkey endogenous FGF23 was evaluated by using the increase in serumphosphorous concentration as an index. The C10 antibody and C15 antibodyproduced in Example 7 were used. Normal cynomolgus monkeys of 2-3 yearsold and body weight 2-3 kg were used as experimental animals. 2 maleanimals and 1 female animal, totaling 3, were used in each group. Thedilution medium used was PBS (−). C10 antibody was prepared at aconcentration of 1 mg/ml and 3 mg/ml, and C15 antibody was prepared at aconcentration of 3 mg/ml. The antibodies were administered once from thesaphenous vein in a volume of 1 mL/kg at a flow rate of about 1 ml/minto achieve a dose of 1 mg/kg and 3 mg/kg for C10 antibody and a dose of3 mg/kg for C15 antibody. Serum phosphorous concentration was measuredusing L type Wako inorganic phosphorous reagent (Wako Pure ChemicalIndustries) and a Hitachi Clinical Analyzer Model 7180 (Hitachi, Ltd.).Blood samples were taken before the administration of antibody, and atday 1, 3, 5, 7, 10, 14, 21 and 28 after the administration of antibody.Measurements of serum phosphorous concentration were conducted for allthe blood sampling points. In the C10 antibody 1 mg/kg group, the C10antibody 3 mg/kg group and the C15 antibody 3 mg/kg group, serumphosphorous concentrations before dosing were 5.37, 5.70 and 5.58 mg/dL,respectively, and there was no difference between groups. In allcynomolgus monkeys, the increase in serum phosphorous concentration wasobserved after the administration. Thus, not only C10 antibody but alsoC15 antibody were shown to have neutralizing activity on cynomolgusmonkey endogenous FGF23. In the C10 antibody 1 mg/kg group, the C10antibody 3 mg/kg group and the C15 antibody 3 mg/kg group, the serumphosphorous concentration 3 days after the administration was 9.03, 9.10and 8.64 mg/dL, respectively. At this time point, the serum phosphorousconcentration in the C10 antibody 1 mg/kg group and the C15 antibody 3mg/kg group reached highest level. On the other hand, the serumphosphorous concentration in the C10 antibody 3 mg/kg group furtherincreased and reached the highest level 5 days after the administration,and the level was 9.75 mg/dL. In the C10 antibody 1 mg/kg group, the C10antibody 3 mg/kg group and the C15 antibody 3 mg/kg group, the maximumdifferences of serum phosphorous concentration between before and afteradministration were 3.67, 4.65 and 3.06 mg/dL, respectively. From thisresult, the effect of C10 antibody on the increase in serum phosphorousconcentration was shown to be higher compared to that of C15 antibody atthe same dose of 3 mg/kg. In addition, surprisingly, C10 antibody at adose of 1 mg/kg increased the serum phosphorous concentration more thanC15 antibody at a dose of 3 mg/kg. Next, the duration of serumphosphorus increase over the pre-dosing level was compared. As a result,the duration of phosphorus increment in the C10 antibody 1 mg/kg group,the C10 antibody 3 mg/kg group and the C15 antibody 3 mg/kg group was14, 28 and 7 days, respectively. From this result, C10 antibody wasshown to have a sustained raising activity of serum phosphorousconcentration compared to that of C15 antibody at the same dose of 3mg/kg. In addition, surprisingly, serum phosphorous concentrationincreased higher at peak and sustained high level much longer by C10antibody at a dose of 1 mg/kg than by C15 antibody at a dose of 3 mg/kg.The above results demonstrate that in cynomolgus monkeys C10 antibodyhas more powerful increasing activity for serum phosphorousconcentration and sustaining activity for serum phosphorousconcentration compared to those of C15 antibody simultaneously obtainedwith C10 antibody. That is, C10 antibody has significantly powerfulneutralizing activity on cynomolgus monkey FGF23 compared to C15antibody.

Example 13 Preparation of Human FGF23 DNA Fragment (SignalSequence-Free)

A reaction solution was prepared by KOD-plus-DNA polymerase (Toyobo),following the manufacturer's instruction. Fifty pmol of FGF23(−SP) FWprimer (SEQ ID NO: 34) and FGF23(−SP) RV primer (SEQ ID NO: 35), andhuman FGF23-cDNA (756 bp from the initiation codon to the stop codon,SEQ ID NO: 36) as the template were added up to 50 μl of the reactionsolution. After incubating the mixture at 94° C. for 3 min, it wassubjected to 30 cycles of a PCR step of heating at 98° C. for 15 sec, at63° C. for 15 sec and at 68° C. for 2 min 30 sec. The mixture was thenincubated at 72° C. for 3 min. The obtained 684 bp amplified fragmentwas separated and collected on a 0.8% gel. The amplified fragment wasrecovered from the collected gel by QIAquick Gel Extraction Kit(Qiagen), following the manufacturer's instruction. The collected PCRamplified fragment was digested with FseI (New England Biolabs Japan),and the enzyme-treated fragment was recovered by QIAquick PCRPurification Kit (Qiagen), following the manufacturer's instruction. Asa result, a partial DNA fragment corresponding to the mature form regionwithout the signal sequence of human FGF23 was obtained.

FGF23(-SP) FW: (SEQ ID NO: 34) TATCCCAATGCCTCCCCACTGCTCGGCTCCAGCTGFGF23(-SP) RV: (SEQ ID NO: 35, including the FseI site)TTGGCCGGCCCTAGATGAACTTGGCGAAGGGGCGGCAGCCTTCCG

The nucleotide sequence of human FGF23 (nucleotides in the signalsequence region are underlined, and nucleotides in the mature formregion excluding the signal sequence region from the full length aresurrounded by a rectangular line.) (SEQ ID NO: 36)

ATGTTGGGGGCCCGCCTCAGGCTCTGGGTCTGTGCCTTGTGCAGCGTCTGCAGCATGAGCGTCCTCAGAGCC

The amino acid sequence of human FGF23 based on SEQ ID NO: 36 as thestandard (amino acid residues in the signal sequence region areunderlined, and amino acid residues in the mature form region excludingthe signal sequence region from the full length are surrounded by arectangular line.) (SEQ ID NO: 37)

MLGARLRLWVCALCSVCSMSVLRA

Example 14 Construction of pPSs FGF23 Vector

pPSs5.5 described in Example 1-8 of WO2006/78072 was digested with SfoIand FseI, and its terminals were subjected to dephosphorylationtreatment with Alkaline Phosphatase derived from E. coli. A DNA fragmentincluding human FGF23 prepared in Example 13 was inserted to the vector.The vector was then introduced into DH5α, and DNA was prepared from theobtained transformants. The nucleotide sequence of the ligated regionwas confirmed to obtain pPSs FGF23 vector (FIG. 10).

Example 15 Construction of pUS FGF23 KI Vector

pCk loxPVΔP described in Example 43-1 of WO2006/78072 was digested withSalI and FseI, and the terminals were subjected to dephosphorylationtreatment with Alkaline Phosphatase derived from E. coli C75. Afterinserting a fragment of about 1.5 kb, wherein the fragment was separatedand collected on a 0.8% agarose gel after digesting pPSs FGF23 vectorprepared in the above Example 14 with SalI and FseI, the vector was thenintroduced into E. coli XL10-Gold Ultracompetent Cells (STRATAGENE). DNAwas prepared from the obtained transformants. The nucleotide sequence ofthe ligated region was confirmed to obtain pUS FGF23 KI vector (FIG.11).

The polynucleotide sequence from the initiation codon to the stop codonof pUS FGF23 KI vector human FGF23 expression unit (985 bp containingmouse Igκ signal sequence including an intron region substituted toFGF23 signal sequence (the underlined part in SEQ ID NO: 38) and FGF23mature form sequence in its downstream, SEQ ID NO: 38) and the aminoacid sequence encoded by the cDNA (247 amino acids, the underlined partrepresents mouse Igκ signal sequence, SEQ ID NO: 39) are shown in thefollowing. Sequence information of mouse Igκ signal sequence includingan intron region was based on MUSIGKVR1 obtained from GenBank (AccessionNo. K02159), and the upstream genome sequence thereof was obtained fromthe UCSC mouse genome database.

SEQ ID NO: 38: ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTGAGAGTGCAGAGAAGTGTTGGATGCAACCTCTGTGGCCATTATGATACTCCATGCCTCTCTGTTCTTGATCACTATAATTAGGGCATTTGTCACTGGTTTTAAGTTTCCCCAGTCCCCTGAATTTTCCATTTTCTCAGAGTGATGTCCAAAATTATTCTTAAAAATTTAAATAAAAAGGTCCTCTGCTGTGAAGGCTTTTATACATATATAACAATAATCTTTGTGTTTATCATTCCAGGTTCCACTGGCTATCCCAATGCCTCCCCACTGCTCGGCTCCAGCTGGGGTGGCCTGATCCACCTGTACACAGCCACAGCCAGGAACAGCTACCACCTGCAGATCCACAAGAATGGCCATGTGGATGGCGCACCCCATCAGACCATCTACAGTGCCCTGATGATCAGATCAGAGGATGCTGGCTTTGTGGTGATTACAGGTGTGATGAGCAGAAGATACCTCTGCATGGATTTCAGAGGCAACATTTTTGGATCACACTATTTCGACCCGGAGAACTGCAGGTTCCAACACCAGACGCTGGAAAACGGGTACGACGTCTACCACTCTCCTCAGTATCACTTCCTGGTCAGTCTGGGCCGGGCGAAGAGAGCCTTCCTGCCAGGCATGAACCCACCCCCGTACTCCCAGTTCCTGTCCCGGAGGAACGAGATCCCCCTAATTCACTTCAACACCCCCATACCACGGCGGCACACCCGGAGCGCCGAGGACGACTCGGAGCGGGACCCCCTGAACGTGCTGAAGCCCCGGGCCCGGATGACCCCGGCCCCGGCCTCCTGTTCACAGGAGCTCCCGAGCGCCGAGGACAACAGCCCGATGGCCAGTGACCCATTAGGGGTGGTCAGGGGCGGTCGAGTGAACACGCACGCTGGGGGAACGGGCCCGGAAGGCTGCCGCCCCTTCGCCAAGTTCATCTAG SEQ ID NO: 39METDTLLLWVLLLWVPGSTGYPNASPLLGSSWGGLIHLYTATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDVYHSPQYHFLVSLGRAKRAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRRHTRSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI

Example 16 Preparation of pUS FGF23 KI Vector for Electroporation

60 μg of pUS FGF23 KI vector was digested at 37° C. for 5 hours usingspermidine-added (1 mM pH7.0, Sigma Aldrich Japan) buffer (RocheDiagnostics, H buffer for restriction enzyme) and NotI (Takara Bio,Inc.). After phenol/chloroform extraction, 2.5 volumes of 100% ethanoland 0.1 volume of 3 M sodium acetate were added, and the mixture waskept at −20° C. for 16 hours. The vector linearized with NotI wascollected by centrifugation and sterilized by adding 70% ethanolthereto. 70% ethanol was removed and air drying was performed for 1 hourin a clean bench. An HBS solution was added to form a 0.5 μg/μL DNAsolution, and the solution was kept at room temperature for 1 hour toprepare pUS FGF23 KI vector for electroporation.

Example 17 Obtaining a PL FGF23 Mouse ES Cell Line using pUS FGF23 KIVector and an RS Element Targeting Mouse ES Cell Line

To obtain a PL FGF23 mouse ES cell line, wherein human FGF23-cDNA wasinserted by homologous recombination into downstream of animmunoglobulin κ light chain gene, according to the method shown inExample 16, pUS FGF23 KI vector linearized with the restriction enzymeNotI was introduced to RS element targeting mouse ES cells according tothe established method (Shinichi Aizawa, “Biotechnology Manual Series 8,Gene Targeting,” Yodosha, 1995). RS element targeting mouse ES cellswere obtained by the method described in Example 10 of WO2006/78072.

The method for culturing RS element targeting mouse ES cells was inaccordance with the described method (Shinichi Aizawa, theaforementioned document), and G418 resistant primary cells in culture(purchased from Invitrogen) treated with mitomycin C (Sigma AldrichJapan) were used as feeder cells. First, the RS element targeting mouseES cells were grown and were treated by trypsin, and suspended in HBS toa density of 3×10⁷ cells/ml. 0.5 ml of the cell suspension was mixedwith 10 μg of vector DNA. Electroporation (Capacitance: 960 μF, voltage:250 V, room temperature) was then performed using Gene Pulser Cuvette(electrode distance: 0.4 cm, Bio Rad Laboratories). The electroporatedcells were suspended in 10 ml of ES culture medium (Shinichi Aizawa, theaforementioned document), and then the cells were seeded to a plasticPetri dish for 100 mm tissue culture (Falcon, Becton Dickinson), whereinfeeder cells were previously seeded. After 36 hours, the culture mediumwas substituted with ES culture medium containing 0.8 μg/ml puromycin(Sigma Aldrich Japan). Colonies which appeared 7 days after were pickedup, and each was grown to confluence in a 24 well plate. Two thirdsthereof were suspended in 0.2 ml of a stock medium (FBS+10% DMSO, SigmaAldrich Japan) and the resulting suspension was kept at −80° C. Theremaining one third was seeded to a 12 well gelatin coated plate. Thecells were cultured for 2 days, and genomic DNA was prepared from 106 to107 cells using Puregene DNA Isolation Kits (Qiagen). The resultinggenomic DNA of puromycin resistant RS element targeting mouse ES cellswas digested with the restriction enzyme EcoRI (Takara Bio, Inc.) andseparated by agarose gel electrophoresis. Subsequently, Southernblotting was performed to detect homologous recombinants by using as theprobe Ck 3′ probe which was the DNA fragment of the 3′ terminal of Iglight chain Jκ-Cκ genomic DNA (XhoI to EcoRI, about 1.4 kb, WO00/10383,FIG. 5) used in the invention described in WO00/10383 (see Example 48).A band (15.1 kb) was detected due to EcoRI digestion in the wild type RSelement targeting mouse ES cells. A new band (12.8 kb) is expected toappear below the band in addition to the band (FIG. 12) in a homologousrecombinant, and the new band was detected in the puromycin resistantstrain. That is, these clones were proven to be having human FGF23-cDNAinserted into downstream of the immunoglobulin κ chain gene in one ofthe alleles.

Example 18 Obtaining a US FGF23 Mouse ES Cell Line by Deleting the DrugResistance Genes from a PL FGF23 Mouse ES Cell Line

To obtain a US FGF23 gene introduced mouse ES cell line, wherein 2 kindsof drug resistance genes (Puror, Neor) were deleted, from a PL FGF23mouse ES cell line, pCAGGS-Cre vector (Sunaga et al., Mol Reprod Dev.,46: 109-113, 1997) was introduced to PL FGF23 mouse ES cells accordingto the established method (Shinichi Aizawa, “Biotechnology Manual Series8, Gene Targeting,” Yodosha, 1995).

The method for culturing PL FGF23 mouse ES cells was in accordance withthe described method (Shinichi Aizawa, the aforementioned document), andG418 resistant primary cells in culture (purchased from Invitrogen)treated with mitomycin C (Sigma Aldrich Japan) were used as feedercells. First, PL FGF23 mouse ES cells were grown and were treated withtrypsin, and suspended in HBS to a density of 3×107 cells/ml. 0.5 ml ofthe cell suspension was mixed with 10 μg of vector DNA. Electroporation(Capacitance: 960 μF, voltage: 250 V, room temperature) was thenperformed using a Gene Pulser Cuvette (electrode distance: 0.4 cm, BioRad Laboratories). The electroporated cells were suspended in 10 ml ofES culture medium (Shinichi Aizawa, the aforementioned document), andthen 2.5 ml of the suspension was seeded to a plastic Petri dish for 60mm tissue culture (Falcon, Becton Dickinson), wherein feeder cells werepreviously seeded. After 30 hours, 1000 cells of the ES cells wereseeded to a plastic Petri dish for 100 mm tissue culture (Falcon, BectonDickinson), wherein feeder cells were previously seeded. Colonies whichappeared 6 days after were picked up, and each was grown to confluencein a 24 well plate. Two thirds thereof were suspended in 0.2 ml of astock medium (FBS+10% DMSO, Sigma Aldrich Japan) and the resultingsuspension was kept at −80° C. The remaining one third was seeded to a12 well gelatin coated plate. The cells were cultured for 2 days, andgenomic DNA was prepared from 106 to 107 cells using Puregene DNAIsolation Kits (Qiagen). The resulting genomic DNA of mouse ES cells wasdigested with the restriction enzyme EcoRI (Takara Bio, Inc.) andseparated by agarose gel electrophoresis. Subsequently, Southernblotting was performed to detect an ES cell line, wherein only the Purorgene between loxPV sequences was deleted, by using as the probe Ck 3′probe which was the DNA fragment of the 3′ terminal of Ig light chainJκ-Cκ genomic DNA (XhoI to EcoRI, about 1.4 kb, WO00/10383, FIG. 5) usedin the invention described in WO00/10383 (see Example 48). Two bands(15.1 kb and 12.8 kb) were detected due to EcoRI digestion in the EScells retaining the Puror gene, and two bands (15.1 kb and 10.9 kb) weredetected due to EcoRI digestion in the ES cell line, wherein only thePuror gene was deleted (FIG. 12). In addition, by using the Southernblotting membrane obtained in the procedure similar to the above, and3′KO-probe prepared by the method shown in Example 9 of WO2006/78072 asthe probe, the ES cell line, wherein the only the Neor gene between loxPsequences was deleted, was detected. Two bands (7.4 K and 5.7 K) weredetected due to EcoRI digestion in the ES cells retaining the Neor gene,and two bands (5.7 K and 4.6 K) were detected due to EcoRI digestion inthe ES cell line, wherein only the Neor gene was deleted (FIG. 12). Fromthese results, the US FGF23 mouse ES cell line, wherein 2 kinds of thedrug resistance genes (Puror, Neor) were deleted simultaneously, wasobtained from the PL FGF23 mouse ES cell line.

Example 19 Preparation of a US FGF23 KI Chimeric Mouse using a US FGF23Mouse ES Cell Line and a Host Embryo Derived from a B LymphocyteDeficient Mouse Strain

In a homozygous knockout for the immunoglobulin μ chain gene, functionalB lymphocytes are deficient and antibodies are not produced (Kitamura etal., Nature, 350: 423-426, 1991). Embryos obtained by cross-breeding theabove individual homozygous male and female grown in a clean environmentwere used as the hosts for preparing chimeric mice in the presentExample. In such case, the majority of functional B lymphocytes in achimeric mouse were derived from the injected ES cells. In the presentExample, an individual immunoglobulin μ chain gene knockout mousedescribed in a report by Tomizuka et al. (Proc. Natl. Acad. Sci. USA,97: 722-7, 2000), which was backcrossed to the MCH (ICR) strain (CLEAJapan, Inc.) 3 times or more, was used for host embryo preparation.

The US FGF23 mouse ES cell line obtained in the above Example 18,wherein the insertion of human FGF23-cDNA downstream of animmunoglobulin κ chain gene was confirmed, was started from a frozenstock, and the cells were injected to a 8-cell stage embryo obtained bycross-breeding individual male and female mice of the aboveimmunoglobulin μ chain gene knockout homozygotes, with 8-10 cells perembryo. After overnight culture in ES culture medium (Shinichi Aizawa,“Biotechnology Manual Series 8, Gene Targeting,” Yodosha, 1995), theembryos were developed into blastocysts. The injection embryos were thentransplanted to the uterus in an adopted parent MCH (ICR) mouse (CLEAJapan, Inc.) 2.5 days after pseudopregnancy treatment, with about 10injection embryos per one side of the uterus, respectively. As a resultof transplanting the injection embryos prepared by using a US FGF23mouse ES cell line prepared in Example 18, chimeric offspring mice wereborn. An individual chimera is determined by the coat color, in whichwhether or not the ES cell-derived wild type color (dark brown) can berecognized in the host embryo-derived white color. Among the chimericoffspring mice born, individual mice obviously having parts in the wildtype color in the coat color, that is, having recognizable contributionof the ES cells, were obtained. From these results, the US FGF23 mouseES cell line, wherein human FGF23-cDNA is inserted into downstream of animmunoglobulin κ chain gene, was shown to maintain chimeric formingability. That is, the cell line has the ability to differentiate intonormal tissues of an individual mouse. In addition, the US FGF23 KIchimeric mouse, as will be described later in Example 21, has a highblood FGF23 concentration, and could be used as an animal model ofdisease exhibiting findings similar to hypophosphatemic rickets.

Example 20 Preparation of Control Chimeric Mouse

A chimeric mouse, in which functional genes including the humanFGF23-cDNA prepared according to the method described in Example 11 ofWO2006/78072 are not inserted, was used as an individual controlchimeric mouse (WT mouse) in the experiment administering C10 antibodyto US FGF23 KI chimeric mouse in the following Example 21.

Example 21 Verification of the Effect of C10 Antibody on Improvement inPathology using a US FGF23 KI Chimeric Mouse

Examples 10 and 12 demonstrated that C10 antibody significantlysuppresses the effect of endogenous FGF23 and elevates the serumphosphorous concentration and serum 1,25D concentration thereof comparedto 2C3B antibody and C15 antibody in normal cynomolgus monkey. It hasbeen strongly suggested that antibody having neutralizing activity onhuman FGF23 has therapeutic effect on human diseases such astumor-induced osteomalacia, hypophosphatemic rickets including XLH andthe like, and osteomalacia which are caused by excessive FGF23.Therefore, the C10 antibody obtained in the present invention wasinvestigated for the effect on improvement in pathology caused byexcessive human FGF23. For the trial of this therapeutic effect of C10antibody, experiments were conducted using a US FGF23 KI chimeric mouse(referred to as an “hFGF23KI mouse” hereinafter) prepared in Example 19.12 hFGF23 KI mice were used as disease-model animals and 6 normalcontrol mice (WT mice, prepared in Example 20) of the same weeks of agewere used as the comparative controls. At 7 weeks of age, serum ofhFGF23 KI mice was collected to measure the serum concentration of FGF23(FGF-23 ELISA KIT, Kainos Laboratories, Inc.) and phosphorus,respectively. Compared to the WT mice, serum FGF23 concentration wassignificantly increased in hFGF23 KI mice (WT mice; n=6, 163 pg/mL,hFGF23KI mice; n=12, 1467 pg/mL). From this result, it was suggestedthat the introduction of the human FGF23 gene to the hFGF23 KI mouse wasprecisely performed and that, in addition, excessive exogenous humanFGF23 was present in the hFGF23 KI mouse blood. In addition, compared tothe WT mice, in hFGF23 KI mice, a significant reduction in the serumphosphorous concentration was shown (WT mice; n=6, 5.82 mg/dL, hFGF23KImice; n=12, 2.62 mg/dL). It was also suggested that hypophosphatemia wasinduced due to excessive human FGF23 action in hFGF23 KI mice. At thistime point, 12 hFGF23 KI mice were divided into the following 2 groupsof 6 mice each, having an equal FGF23 concentration: the C10 antibodyadministered group and the control IgG1 administered group (FIG. 13).Next, since 8 weeks of age, repeated intravenous administration of C10antibody or purified human IgG1 (control antibody) for isotype controlwas conducted at a dose of 30 mg/kg and frequency of once a week fivetimes. Blood samples were taken before the first administration and 3days after the administration, and the serum was obtained. Appendiculargrip strength was measured 24 hours after the fourth administrationusing a Saitoh-GRIP STRENGTH METER (MK-380S, Muromachi Kikai Co., Ltd.).Appendicular grip strength was evaluated by using as an index themaximum force (grip strength) exerted by a mouse, wherein the mouse wasplaced on a measurement grid, to let the mouse grip the grid, and thenthe mouse was pulled by the tail horizontally by our hand until theanimal released the grid for being unable to bear the withdrawing force.Bones were evaluated 24 hours after the fifth administration. Thecollected femur and tibia from mice euthanized by blood drawing from theheart under anesthesia were fixed in 70% ethanol. Serum phosphorousconcentration was measured at the before first administration, 3 daysafter the first administration and 24 hours after the fifthadministration. Undecalcified femur was embedded in resin, and stainedwith Villanueva-Goldner for histological evaluation. Bone mineralcontent in tibia was measured through the ashing process.

As a result, significantly low serum phosphorus concentration wasobserved in the hFGF23KI mouse control antibody administered group atthe time of grouping and 24 hours after fifth administration compared tothe WT mouse control antibody administered group, which means continuoushypophosphatemic conditions (FIG. 14). On the other hand, it wasobserved that the serum phosphorous concentration at 3 days afteradministration was increased in hFGF23KI mouse C10 antibody administeredgroup to the same level as that in the WT mouse control antibodyadministered group (FIG. 14). In addition, the serum phosphorousconcentration after the fifth administration in the hFGF23KI mouse C10antibody administered group was also the same level as that in the WTmouse control antibody administered group, which means the effect of C10antibody for the increment of serum phosphorus concentration wasmaintained even after five times of administration (FIG. 15).

As a case of hypophosphatemic patients, skeletal muscle weakness hasbeen reported (Baker and Worthley, Crit. Care Resusc., 4: 307-315,2000). In the present study, hFGF23KI mice had been expected the muscleweakness because of the hypophosphatemia. Consequently, appendiculargrip strength was measured by the above method as an index of muscleweakness, and compared among groups. As a result, the grip strength ofthe hFGF23KI mouse control antibody administered group was shown to besignificantly low compared to that of the WT mouse control antibodyadministered group, and muscle weakness was observed in this diseasemodel (FIG. 16). In contrast, significant improvement of grip strengthwas observed in the hFGF23KI mouse C10 antibody administered group (FIG.16).

Next, under-calcified femoral tissues were stained by Villanueva-Goldnermethod for histological observation. As a result, a large amount ofosteoid (shown in red in FIG. 17) was observed in the bone in thehFGF23KI mouse control antibody administered group compared to that inWT mouse control antibody administered group, suggesting thatcalcification defect was induced in that group. This is widely known asa characteristic symptom of rickets. In contrast, in the hFGF23KI micereceived C10 antibody treatment, reduction of the area occupied withosteoid was observed, and predicted that osteoid was replaced withcalcified bones (shown in green in FIG. 17). From this result, it wassuggested that C10 antibody improves bone calcification reduced byexcessive FGF23. Consequently, the amount of minerals contained in tibiawas measured by calcification, and compared between each group. Theamount of minerals contained in tibia in the hFGF23KI mouse controlantibody administered group was significantly reduced compared to the WTmouse control antibody administered group (FIG. 18). In contrast, in thehFGF23KI mouse C10 antibody administered group, improvement in theamount of minerals was confirmed (FIG. 18). From the above results, itwas confirmed that, in hFGF23KI mice, C10 antibody administrationneutralizes the effect of excessively acting human FGF23 in vivo, andimproves various symptoms of hypophosphatemic rickets such ashypophosphatemia, muscle weakness, bone calcification disorder and thelike. That is, C10 antibody was shown to be an effective therapeuticagent for various human diseases involving FGF23.

INDUSTRIAL APPLICABILITY

The C10 antibody of the present invention which is an antibody againstFGF23 has high activity to raise serum phosphate concentrations in vivoin a sustained manner and/or to raise serum 1,25D concentrations in asustained manner as compared to known antibodies against FGF23. Thepresent invention can be used with dramatic effects as an agent forprevention or treatment of diseases which are caused by excessive actionof FGF23 or for diseases which may be improved in the pathology bycontrolling the action of FGF23.

What is claimed is:
 1. An antibody or functional fragment thereof that binds to FGF23, wherein the antibody or functional fragment comprises a heavy and light chain, said heavy chain comprises CDR1 shown by SEQ ID NO: 40, CDR2 shown by SEQ ID NO: 41, and CDR3 shown by SEQ ID NO: 42, and said light chain comprises CDR1 shown by SEQ ID NO: 43, CDR2 shown by SEQ ID NO: 44, and CDR3 shown by SEQ ID NO:
 45. 